Novel primers and uses thereof

ABSTRACT

Disclosed here is a composition comprising a primer that is (a) a loopable primer comprising a target-specific section, an adaptor section, and a stem-forming section, wherein the stem-forming section is hybridizable to a portion of the target-specific section to form a stem structure, or (b) a split primer comprising a first target-specific section, a second target-specific section, and an adaptor section positioned between the first target-specific section and the second target-specific section, or (c) a split-loopable primer comprising a first target-specific section, a second target-specific section, a stem-forming section positioned between the first target-specific section and the second target-specific section, and an adaptor section, or comprising a first adaptor section, a second adaptor section, a stem-forming section positioned between the first adaptor section and the second adaptor section, and a target-specific section. Also disclosed is a method for amplifying a target locus of interest from a template DNA, comprising at least two pre-amplification cycles using the loopable primer, the split primer and/or the split-loopable primer, wherein each amplification cycle comprises annealing the primer to the template DNA or pre-amplification product thereof and elongating the annealed primer. Further disclosed is a kit for amplifying a target locus of interest, comprising the loopable primer, the split primer, and/or the split-loopable primer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/617,066, filed Jan. 12, 2018, which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 27, 2018, isnamed N_022_WO_01_SL.txt and is 3,844 bytes in size.

BACKGROUND

Molecular barcodes or indexing sequences have been used in nextgeneration sequencing to reduce quantitative bias introduced byreplication, by tagging each nucleic acid fragment with a molecularbarcode or indexing sequence. Sequence reads that have differentmolecular barcodes or indexing sequences represent different originalnucleic acid molecules. By referencing the molecular barcodes orindexing sequences, PCR artifacts, such as sequence changes generated bypolymerase errors that are not present in the original nucleic acidmolecules can be identified and separated from real variants/mutationspresent in the original nucleic acid molecules.

However, in order to apply molecular barcodes or indexing sequences inhighly multiplex PCR, a need exists for suppressing primer dimersformation, avoiding barcode resampling, and reducing nonspecific primerbinding and formation of primer concatamers.

SUMMARY

The present inventions are directed to compositions, methods, and kitsfor amplification of nucleic acids. In a first aspect, the inventionsdescribed herein relate to a composition comprising a primer that is:(a) a loopable primer comprising a target-specific section, an adaptorsection, and a stem-forming section, wherein the stem-forming section ishybridizable to a portion of the target-specific section to form a stemstructure, or (b) a split primer comprising a first target-specificsection, a second target-specific section, and an adaptor sectionpositioned between the first target-specific section and the secondtarget-specific section, or (c) a split-loopable primer comprising afirst target-specific section, a second target-specific section, and astem-forming section positioned between the first target-specificsection and the second target-specific section, and an adaptor section,or comprising a first adaptor section, a second adaptor section, and astem-forming section positioned between the first adaptor section andthe second adaptor section, and a target-specific section.

In a second aspect, the inventions described herein relate to a methodfor amplifying a target locus of interest from a template DNA,comprising at least two pre-amplification cycles using a primer that is:(a) a loopable primer comprising a target-specific section, an adaptorsection, and a stem-forming section, wherein the stem-forming section ishybridizable to a portion of the target-specific section to form a stemstructure, or (b) a split primer comprising a first target-specificsection, a second target-specific section, and an adaptor sectionpositioned between the first target-specific section and the secondtarget-specific section, or (c) a split-loopable primer comprising afirst target-specific section, a second target-specific section, and astem-forming section positioned between the first target-specificsection and the second target-specific section, and an adaptor section,or comprising a first adaptor section, a second adaptor section, and astem-forming section positioned between the first adaptor section andthe second adaptor section, and a target-specific section; wherein eachpre-amplification cycle comprises annealing the primer to the templateDNA or pre-amplification product thereof and elongating the annealedprimer.

In a third aspect, the inventions described herein relate to a kit foramplifying a target locus of interest, comprising a primer that is: (a)a loopable primer comprising a target-specific section, an adaptorsection, and a stem-forming section, wherein the stem-forming section ishybridizable to a portion of the target-specific section to form a stemstructure, or (b) a split primer comprising a first target-specificsection, a second target-specific section, and an adaptor sectionpositioned between the first target-specific section and the secondtarget-specific section, or (c) a split-loopable primer comprising afirst target-specific section, a second target-specific section, and astem-forming section positioned between the first target-specificsection and the second target-specific section, and an adaptor section,or comprising a first adaptor section, a second adaptor section, and astem-forming section positioned between the first adaptor section andthe second adaptor section, and a target-specific section.

In a fourth aspect, the inventions described herein relate to a methodfor determining copy number variation of a target locus of interest,comprising: pre-amplifying the target locus of interest from a templateDNA using at least two pre-amplification cycles with: (a) one or moreloopable primers each comprising a target-specific section, an adaptorsection, a molecular indexing section, and a stem-forming section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the adaptor section and the molecular indexing sectionand the 5′-portion of the target-specific section, wherein the adaptorsection comprises a universal adaptor sequence for PCR amplification,and wherein the molecular indexing section comprises a molecule indexingsequence, (b) one or more split-loopable primers each comprising a firsttarget-specific section, a second target-specific section, astem-forming section positioned between the first target-specificsection and the second target-specific section, a molecular indexingsection, and an adaptor section, wherein the stem-forming section ishybridizable to a portion of the second target-specific section to forma stem structure and a loop comprising the adaptor section and themolecular indexing section, wherein the adaptor section comprises auniversal adaptor sequence for PCR amplification, and wherein themolecular indexing section comprises a molecule indexing sequence, or(c) one or more split-loopable primers each comprising a first adaptorsection, a second adaptor section, a stem-forming section positionedbetween the first adaptor section and the second adaptor section, amolecular indexing section, and a target-specific section, wherein thetarget-specific section comprises a 5′-portion and a 3′-portion and thestem-forming section is hybridizable to the 3′-portion of thetarget-specific section to form a stem structure and a loop comprisingthe second adaptor section and the molecular indexing section and the5′-portion of the target-specific section, wherein the first and/orsecond adaptor sections comprise a universal adaptor sequence for PCRamplification, and wherein the molecular indexing section comprises amolecule indexing sequence; amplifying the pre-amplification productusing one or more PCR primers hybridizable to the universal adaptorsequence; and sequencing the amplification product to determine copynumber variation of the target locus of interest using the moleculeindexing sequence.

In a fifth aspect, the inventions described herein relate to a methodfor determining fetal aneuploidy, comprising: pre-amplifying a pluralityof target loci of interest of one or more chromosomes from cell-free DNAisolated from a maternal blood sample, using at least twopre-amplification cycles with: (a) a plurality of loopable primers eachcomprising a target-specific section, an adaptor section, a molecularindexing section, and a stem-forming section, wherein thetarget-specific section comprises a 5′-portion and a 3′-portion and thestem-forming section is hybridizable to the 3′-portion of thetarget-specific section to form a stem structure and a loop comprisingthe adaptor section and the molecular indexing section and the5′-portion of the target-specific section, wherein the adaptor sectioncomprises a universal adaptor sequence for PCR amplification, andwherein the molecular indexing section comprises a molecule indexingsequence, (b) a plurality of split-loopable primers each comprising afirst target-specific section, a second target-specific section, astem-forming section positioned between the first target-specificsection and the second target-specific section, a molecular indexingsection, and an adaptor section, wherein the stem-forming section ishybridizable to a portion of the second target-specific section to forma stem structure and a loop comprising the adaptor section and themolecular indexing section, wherein the adaptor section comprises auniversal adaptor sequence for PCR amplification, and wherein themolecular indexing section comprises a molecule indexing sequence, or(c) a plurality of split-loopable primers each comprising a firstadaptor section, a second adaptor section, a stem-forming sectionpositioned between the first adaptor section and the second adaptorsection, a molecular indexing section, and a target-specific section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the second adaptor section and the molecular indexingsection and the 5′-portion of the target-specific section, wherein thefirst and/or second adaptor sections comprise a universal adaptorsequence for PCR amplification, and wherein the molecular indexingsection comprises a molecule indexing sequence; amplifying thepre-amplification product using one or more PCR primers hybridizable tothe universal adaptor sequence; and sequencing the amplification productto determine fetal aneuploidy using the molecule indexing sequence.

In a sixth aspect, the inventions described herein relate to a methodfor multiplex amplification, comprising: pre-amplifying one or moretarget loci of interest from a template DNA using at least twopre-amplification cycles with: (a) at least a first loopable primer anda second loopable primer each comprising a target-specific section, anadaptor section, and a stem-forming section, wherein the target-specificsection comprises a 5′-portion and a 3′-portion and the stem-formingsection is hybridizable to the 3′-portion of the target-specific sectionto form a stem structure and a loop comprising the adaptor section andthe 5′-portion of the target-specific section, wherein the adaptorsection comprises a universal adaptor sequence for PCR amplification,(b) at least a first split-loopable primer and a second split-loopableprimer each comprising a first target-specific section, a secondtarget-specific section, a stem-forming section positioned between thefirst target-specific section and the second target-specific section,and an adaptor section, wherein the stem-forming section is hybridizableto a portion of the second target-specific section to form a stemstructure and a loop comprising the adaptor section, wherein the adaptorsection comprises a universal adaptor sequence for PCR amplification, or(c) at least a first split-loopable primer and a second split-loopableprimer each comprising a first adaptor section, a second adaptorsection, a stem-forming section positioned between the first adaptorsection and the second adaptor section, and a target-specific section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the second adaptor section and the 5′-portion of thetarget-specific section, wherein the first and/or second adaptorsections comprise a universal adaptor sequence for PCR amplification;and wherein the first primer and the second primer comprisecomplementary sequences in their target-specific sections and arecapable of forming a primer dimer absent protection by the stem-formingsection; and amplifying the pre-amplification product using one or morePCR primers hybridizable to the universal adaptor sequence. Theprevention of primer-dimer formation is particularly useful for PCRtiling (e.g., amplifying overlapping or tiled target sequences in asingle multiplex PCR reaction).

In a seventh aspect, the inventions described herein relate to a methodfor allele-specific amplification, comprising: pre-amplifying one ormore target loci of interest from a template DNA using at least twopre-amplification cycles with: (a) a loopable primer comprising atarget-specific section, an adaptor section, and a stem-forming section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the adaptor section and the 5′-portion of thetarget-specific section, wherein the adaptor section comprises auniversal adaptor sequence for PCR amplification, and wherein theloopable primer comprises an SNV or SNP allele in the 5′- or 3′-portionof the target-specific section, (b) a split-loopable primer comprising afirst target-specific section, a second target-specific section, astem-forming section positioned between the first target-specificsection and the second target-specific section, and an adaptor section,wherein the stem-forming section is hybridizable to a portion of thesecond target-specific section to form a stem structure and a loopcomprising the adaptor section, wherein the adaptor section comprises auniversal adaptor sequence for PCR amplification, and wherein thesplit-loopable primer comprises an SNV or SNP allele in thetarget-specific section, or (c) a split-loopable primer comprising afirst adaptor section, a second adaptor section, a stem-forming sectionpositioned between the first adaptor section and the second adaptorsection, and a target-specific section, wherein the target-specificsection comprises a 5′-portion and a 3′-portion and the stem-formingsection is hybridizable to the 3′-portion of the target-specific sectionto form a stem structure and a loop comprising the second adaptorsection and the 5′-portion of the target-specific section, wherein thefirst and/or second adaptor sections comprise a universal adaptorsequence for PCR amplification, and wherein the split-loopable primercomprises an SNV or SNP allele in the 3′-portion of the target-specificsection; and amplifying the pre-amplification product using one or morePCR primers hybridizable to the universal adaptor sequence.

In an eighth aspect, the inventions described herein relate to a methodfor allele-specific quantitative PCR (qPCR), comprising: pre-amplifyingone or more target loci of interest from a template DNA using at leasttwo pre-amplification cycles with: (a) at least a first loopable primerand a second loopable primer each comprising a target-specific section,an adaptor section, and a stem-forming section, wherein thetarget-specific section comprises a 5′-portion and a 3′-portion and thestem-forming section is hybridizable to the 3′-portion of thetarget-specific section to form a stem structure and a loop comprisingthe adaptor section and the 5′-portion of the target-specific section,wherein the adaptor section of the first loopable primer comprises auniversal adaptor sequence for PCR amplification and a firstprobe-specific sequence capable of binding to a first fluorescent probe,wherein the adaptor section of the second loopable primer comprises auniversal adaptor sequence for PCR amplification and a secondprobe-specific sequence capable of binding to a second fluorescentprobe, wherein the 5′- or 3′-portion of the target-specific section ofthe first loopable primer comprises a first SNV or SNP allele, andwherein the 5′- or 3′-portion of the target-specific section of thesecond loopable primer comprises a second SNV or SNP allele, (b) atleast a first split-loopable primer and a second split-loopable primereach comprising a first target-specific section, a secondtarget-specific section, a stem-forming section positioned between thefirst target-specific section and the second target-specific section,and an adaptor section, wherein the stem-forming section is hybridizableto a portion of the second target-specific section to form a stemstructure and a loop comprising the adaptor section, wherein the adaptorsection of the first split-loopable primer comprises a universal adaptorsequence for PCR amplification and a first probe-specific sequencecapable of binding to a first fluorescent probe, wherein the adaptorsection of the second split-loopable primer comprises a universaladaptor sequence for PCR amplification and a second probe-specificsequence capable of binding to a second fluorescent probe, wherein thetarget-specific section of the first split-loopable primer comprises afirst SNV or SNP allele, and wherein the target-specific section of thesecond split-loopable primer comprises a second SNV or SNP allele, or(c) at least a first split-loopable primer and a second split-loopableprimer each comprising a first adaptor section, a second adaptorsection, a stem-forming section positioned between the first adaptorsection and the second adaptor section, and a target-specific section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the second adaptor section and the 5′-portion of thetarget-specific section, wherein the first and/or second adaptorsections of the first split-loopable primer comprise a universal adaptorsequence for PCR amplification and a first probe-specific sequencecapable of binding to a first fluorescent probe, wherein the firstand/or second adaptor sections of the second split-loopable primercomprise a universal adaptor sequence for PCR amplification and a secondprobe-specific sequence capable of binding to a second fluorescentprobe, wherein the 5′- or 3′-portion of the target-specific section ofthe first split-loopable primer comprises a first SNV or SNP allele, andwherein the 5′- or 3′-portion of the target-specific section of thesecond split-loopable primer comprises a second SNV or SNP allele;amplifying the pre-amplification product using one or more PCR primershybridizable to the universal adaptor sequence in the presence of thefirst fluorescent probe and the second fluorescent probe; and detectingreal-time intensity of fluorescent signal from the first fluorescentprobe and the second fluorescent probe. Alternatively, the method forallele-specific qPCR does not require a pre-amplification step, andinstead comprises amplifying one or more target loci of interest from atemplate DNA using the primer of (a), (b) or (c) in the presence of thefirst fluorescent probe and the second fluorescent probe; and detectingreal-time intensity of fluorescent signal from the first fluorescentprobe and the second fluorescent probe.

In a ninth aspect, the inventions described herein relate to a methodfor allele-specific digital PCR (dPCR), comprising: pre-amplifying oneor more target loci of interest from a template DNA using at least twopre-amplification cycles with: (a) at least a first loopable primer anda second loopable primer each comprising a target-specific section, anadaptor section, and a stem-forming section, wherein the target-specificsection comprises a 5′-portion and a 3′-portion and the stem-formingsection is hybridizable to the 3′-portion of the target-specific sectionto form a stem structure and a loop comprising the adaptor section andthe 5′-portion of the target-specific section, wherein the adaptorsection of the first loopable primer comprises a universal adaptorsequence for PCR amplification and a first probe-specific sequencecapable of binding to a first fluorescent probe, wherein the adaptorsection of the second loopable primer comprises a universal adaptorsequence for PCR amplification and a second probe-specific sequencecapable of binding to a second fluorescent probe, wherein the 5′- or3′-portion of the target-specific section of the first loopable primercomprises a first SNV or SNP allele, and wherein the 5′- or 3′-portionof the target-specific section of the second loopable primer comprises asecond SNV or SNP allele, (b) at least a first split-loopable primer anda second split-loopable primer each comprising a first target-specificsection, a second target-specific section, a stem-forming sectionpositioned between the first target-specific section and the secondtarget-specific section, and an adaptor section, wherein thestem-forming section is hybridizable to a portion of the secondtarget-specific section to form a stem structure and a loop comprisingthe adaptor section, wherein the adaptor section of the firstsplit-loopable primer comprises a universal adaptor sequence for PCRamplification and a first probe-specific sequence capable of binding toa first fluorescent probe, wherein the adaptor section of the secondsplit-loopable primer comprises a universal adaptor sequence for PCRamplification and a second probe-specific sequence capable of binding toa second fluorescent probe, wherein the target-specific section of thefirst split-loopable primer comprises a first SNV or SNP allele, andwherein the target-specific section of the second split-loopable primercomprises a second SNV or SNP allele, or (c) at least a firstsplit-loopable primer and a second split-loopable primer each comprisinga first adaptor section, a second adaptor section, a stem-formingsection positioned between the first adaptor section and the secondadaptor section, and a target-specific section, wherein thetarget-specific section comprises a 5′-portion and a 3′-portion and thestem-forming section is hybridizable to the 3′-portion of thetarget-specific section to form a stem structure and a loop comprisingthe second adaptor section and the 5′-portion of the target-specificsection, wherein the first and/or second adaptor sections of the firstsplit-loopable primer comprise a universal adaptor sequence for PCRamplification and a first probe-specific sequence capable of binding toa first fluorescent probe, wherein the first and/or second adaptorsections of the second split-loopable primer comprise a universaladaptor sequence for PCR amplification and a second probe-specificsequence capable of binding to a second fluorescent probe, wherein the5′- or 3′-portion of the target-specific section of the firstsplit-loopable primer comprises a first SNV or SNP allele, and whereinthe 5′- or 3′-portion of the target-specific section of the secondsplit-loopable primer comprises a second SNV or SNP allele; partitioningthe pre-amplification product into a plurality of reaction volumes;amplifying the pre-amplification product in each reaction volume usingone or more PCR primers hybridizable to the universal adaptor sequencein the presence of the first fluorescent probe and the secondfluorescent probe; and detecting presence or absence of fluorescentsignal from the first fluorescent probe and the second fluorescentprobe. Alternatively, the method for allele-specific dPCR does notrequire a pre-amplification step, and instead comprises partitioning asample into a plurality of reaction volumes; amplifying one or moretarget loci of interest from a template DNA in each reaction volumeusing the primer of (a), (b) or (c) in the presence of the firstfluorescent probe and the second fluorescent probe; and detectingpresence or absence of fluorescent signal from the first fluorescentprobe and the second fluorescent probe.

These and other features, together with the organization and manner ofoperation thereof, will become apparent from the following detaileddescription when taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows some embodiments of the primers described herein (Scheme Ato C and Control Scheme).

FIG. 2 shows one embodiment of a loopable primer in which a 3′-endportion of the target-specific section is configured to form a stemstructure with a complementary sequence.

FIG. 3 shows one embodiment of a loopable primer in which a 5′-endportion of the target-specific section is configured to form a stemstructure with a complementary sequence.

FIG. 4 shows one embodiment of a split primer in which thetarget-specific section is split into a 5′-end portion and a 3′-endportion that are separated by an adaptor sequence.

FIG. 5 shows workflow of an example amplification process including 2pre-amplification cycles using the primers described herein.

FIG. 6 shows on-target rates of the example amplification processincluding 2 pre-amplification cycles using the primers described herein.

FIG. 7 shows workflow of an example amplification process including 3 or10 pre-amplification cycles using the primers described herein.

FIG. 8 shows on-target rates of the example amplification processincluding 3 or 10 pre-amplification cycles using the primers describedherein.

FIG. 9 shows consistent MIT counts between replicate samples (Scheme A,2 pre-amplification cycles).

FIG. 10 shows primer and product sequences according to one embodimentof a loopable primer, which includes 2 mismatched nt in the loopableprimer.

FIG. 11 shows primer and product sequences according to one embodimentof a loopable primer.

FIG. 12 shows primer and product sequences according to one embodimentof a split primer.

FIG. 13 shows some embodiments of the split-loopable primers describedherein (Scheme D to E).

FIG. 14 shows consistent MIT counts between replicate samples in highlymultiplex PCR (Scheme A, 2 pre-amplification cycles, workflow as FIG.5). The on-target rate of this example amplification process is 83%.

DETAILED DESCRIPTION

Reference will now be made in detail to some specific embodiments of theinvention contemplated by the inventors for carrying out the invention.Certain examples of these specific embodiments are illustrated in theaccompanying drawings. While the invention is described in conjunctionwith these specific embodiments, it will be understood that it is notintended to limit the invention to the described embodiments. On thecontrary, it is intended to cover alternatives, modifications, andequivalents as may be included within the spirit and scope of theinvention as defined by the appended claims.

In the following description, numerous specific details are set forth inorder to provide a thorough understanding of the present invention.Particular example embodiments of the present invention may beimplemented without some or all of these specific details.

Various techniques and mechanisms of the present invention willsometimes be described in singular form for clarity. However, it shouldbe noted that some embodiments include multiple iterations of atechnique or multiple instantiations of a mechanism unless notedotherwise.

The disclosure of the following patent applications are incorporatedherein by reference: International patent application No.PCT/US2006/045281 titled “SYSTEM AND METHOD FOR CLEANING NOISY GENETICDATA AND USING DATA TO MAKE PREDICTIONS”; International patentapplication No. PCT/US2008/003547 titled “SYSTEM AND METHOD FOR CLEANINGNOISY GENETIC DATA AND DETERMINING CHROMSOME COPY NUMBER”; Internationalpatent application No. PCT/US2009/034506 titled “METHODS FOR CELLGENOTYPING”; International patent application No. PCT/US2009/045335titled “METHODS FOR EMBRYO CHARACTERIZATION AND COMPARISON”;International patent application No. PCT/US2009/052730 titled “METHODSFOR ALLELE CALLING AND PLOIDY CALLING”; International patent applicationNo. PCT/US2010/050824 titled “METHODS FOR NON-INVASIVE PRENATAL PLOIDYCALLING”; International patent application No. PCT/US2011/037018 titled“METHODS FOR NON-INVASIVE PRENATAL PLOIDY CALLING”; International patentapplication No. PCT/US2011/061506 titled “METHODS FOR NON-INVASIVEPRENATAL PLOIDY CALLING”; International patent application No.PCT/US2011/066938 titled “METHODS FOR NON-INVASIVE PRENATAL PATERNITYTESTING”; International patent application No. PCT/US2012/066339 titled“HIGHLY MULTIPLEX PCR METHODS AND COMPOSITIONS”; International patentapplication No. PCT/US2013/055205 titled “METHODS AND COMPOSITIONS FORREDUCING GENETIC LIBRARY CONTAMINATION”; International patentapplication No. PCT/US2013/057924 titled “METHODS FOR INCREASING FETALFRACTION IN MATERNAL BLOOD”; International patent application No.PCT/US2014/051926 titled “METHODS OF USING LOW FETAL FRACTIONDETECTION”; International patent application No. PCT/US2014/057843titled “PRENATAL DIAGNOSTIC RESTING STANDARDS”; International patentapplication No. PCT/US2015/026957 titled “DETECTING MUTATIONS AND PLOIDYIN CHROMOSOMAL SEGMENTS”; International patent application No.PCT/US2016/031686 titled “METHODS AND COMPOSITIONS FOR DETERMININGPLOIDY”; and U.S. patent application Ser. No. 15/372,279 titled“COMPOSITIONS AND METHODS FOR IDENTIFYING NULCEIC ACID MOLECULES.”

Loopable Primer

Many embodiments of the invention described herein relate to a loopableprimer comprising a target-specific section, an adaptor section, and astem-forming section, wherein the stem-forming section is hybridizableto a portion of the target-specific section to form a stem structure,and wherein the target-specific section is hybridizable to a targetsequence of a template DNA intended for amplification.

In some embodiments, the adaptor section is positioned between thetarget-specific section and the stem-forming section, whereinhybridization between the stem-forming section and the portion of thetarget-specific section forms a loop that comprises the adaptor section.The adaptor section can be, for example, positioned at 5′ side of thetarget-specific section and 3′ side of the stem-forming section. In someembodiments, the adaptor section comprises a universal adaptor sequencefor PCR amplification and/or sequencing.

In some embodiments, the loopable primer further comprises a molecularindexing section comprising a molecule indexing sequence. Molecularindexing sequences, molecular index tags (MIT), or unique identifier(UID) sequences, are described in Kinde et al., PNAS 108(23):9530-9535(2011), as well as in U.S. patent application Ser. No. 15/372,279 titled“COMPOSITIONS AND METHODS FOR IDENTIFYING NULCEIC ACID MOLECULES,” eachof which is incorporated herein by reference in its entirety. In someembodiments, the length of each molecular indexing sequence is about1-20 bp, or about 2-15 bp, or about 3-10 bp, or about 4-8 bp. When botha forward loopable primer and a reverse loopable primer according to theinvention described herein are used for amplifying a target locus ofinterests, the amplification product can include two molecular indexingsequences, the combination of which allows even more accurate molecularcounting than the use of a single molecular indexing sequence. In oneembodiment, the molecule indexing sequence on each loopable primer is aunique molecule indexing sequence. In another embodiment, thecombination of molecule indexing sequences on each pair of loopableforward and reverse primers are unique.

The molecular indexing section can be, for example, positioned betweenthe target-specific section and the adaptor section. The molecularindexing section can be, for example, positioned at 5′ side of thetarget-specific section and 3′ side of the adaptor section. In someembodiments, hybridization between the stem-forming section and theportion of the target-specific section forms a loop that comprises theadaptor section and the molecular indexing section.

In some embodiments, the loopable primer described herein excludesprimers in which the target-specific section does not form part of thestem structure.

Because the loopable primers described herein hides/protects theuniversal adaptor sequences and the molecular indexing sequences, aswell as at least part of the target-specific sequences, the loopableprimers are capable of significantly improving assay specificity bysuppressing primer dimers and non-specific binding in highly multiplexPCR.

Scheme A: FIG. 2 shows one scheme of the loopable primer in which thetarget-specific section comprises a 5′-portion and a 3′-portion, whereinthe stem-forming section is hybridizable to the 3′-portion of thetarget-specific section to form a stem structure. When the 3′-portion ofthe target-specific section is protected in the stem structure, the5′-portion of the target-specific section is available for initiatingtarget hybridization.

In some embodiments, hybridization between the stem-forming section andthe 3′-portion of the target-specific section forms a loop thatcomprises the adaptor section and the 5′-portion of the target-specificsection. The stem loop is designed to protect the molecule indexingsequence and the adaptor sequence from spurious interactions. The stemin the 3′-end also prevents primers from non-target specific extension(i.e. primer dimers).

In some embodiments, the loopable primer further comprises one or moremismatched nucleotides at 3′-terminus of the target-specific sectionthat are not hybridizable to the stem-forming section. In someembodiments, the loopable primer comprises 1, 2, 3, 4, or 5 mismatchednucleotides at 3′-terminus to prevent A-tailing. Alternatively oradditionally, the 5′-terminus of the loopable primer may comprise one ormore mismatched nucleotides that are not hybridizable to thetarget-specific section. In some embodiments, the loopable primercomprises 1, 2, 3, 4, or 5 mismatched nucleotides at 5′-terminus.

As shown in FIG. 10, a preferred embodiment of the loopable primeraccording to Scheme A comprises, from 5′ to 3′, one or more mismatchednucleotides, a stem-forming section, an adaptor section, a molecularindexing section, and a target-specific section, wherein thestem-forming section is the reverse complement of the 3′-portion of thetarget-specific section.

In some embodiments, the size of the stem structure formed between thestem-forming section and the 3′-portion of the target-specific sectionis about 5-20 bp, or about 5-10 bp, or about 10-15 bp, or about 15-20bp.

In some embodiments, the loopable primer according to Scheme A has apreferred annealing temperature for PCR reaction and a meltingtemperature, wherein the stem-forming section and the 3′-portion of thetarget-specific section form the stem structure at the preferredannealing temperature or below and do not form the stem structure at themelting temperature or above. In some embodiments, the preferredannealing temperature is 60° C. or less, 59° C. or less, or 58° C. orless, or 57° C. or less, or 56° C. or less, or 55° C. or less, 54° C. orless, or 53° C. or less, or 52° C. or less, or 51° C. or less, or 50° C.or less. In some embodiments, the melting temperature is 60° C. orabove, or 61° C. or above, or 62° C. or above, or 63° C. or above, or64° C. or above, or 65° C. or above, 66° C. or above, or 67° C. orabove, or 68° C. or above, or 69° C. or above, or 70° C. or above. Insome embodiments, extreme annealing temperatures may be useful, such as30° C. to 80° C.

Scheme B: FIG. 3 shows another scheme of the loopable primer in whichthe target-specific section comprises a 5′-portion and a 3′-portion,wherein the stem-forming section is hybridizable to the 5′-portion ofthe target-specific section to form a stem structure. When the5′-portion of the target-specific section is protected in the stemstructure, the 3′-portion of the target-specific section is availablefor initiating target hybridization

In some embodiments, hybridization between the stem-forming section andthe 5′-portion of the target-specific section forms a loop thatcomprises the adaptor section but not the 3′-end portion of thetarget-specific section. The stem loop is designed to protect themolecule indexing sequence and the adaptor sequence from spuriousinteractions.

In some embodiments, the loopable primer further comprises one or moreof G/C nucleotides positioned at the 5′-side of the target-specificsection for stabilizing the stem structure with one or morecomplementary G/C nucleotides positioned at the 3′-side of thestem-forming section. In some embodiments, the loopable primer comprises1, 2, 3, 4, or 5 G/C nucleotides positioned at the 5′-side of thetarget-specific section (i.e., at the neck of the stem loop).

As shown in FIG. 11, a preferred embodiment of the loopable primeraccording to Scheme B comprises, from 5′ to 3′, a stem-forming section,one or more G/C nucleotides, an adaptor section, a molecular indexingsection, one or more G/C nucleotides, and a target-specific section,wherein the stem-forming section is the reverse complement of the5′-portion of the target-specific section.

In some embodiments, the size of the stem structure formed between thestem-forming section and the 5′-portion of the target-specific sectionis about 5-20 bp, or about 5-10 bp, or about 10-15 bp, or about 15-20bp.

In some embodiments, the loopable primer according to Scheme B has apreferred annealing temperature and a melting temperature for PCRreaction, wherein the stem-forming section and the 5′-portion of thetarget-specific section form the stem structure at the preferredannealing temperature or below and do not form the stem structure at themelting temperature or above. In some embodiments, the preferredannealing temperature is 60° C. or less, 59° C. or less, or 58° C. orless, or 57° C. or less, or 56° C. or less, or 55° C. or less, 54° C. orless, or 53° C. or less, or 52° C. or less, or 51° C. or less, or 50° C.or less. In some embodiments, the melting temperature is 60° C. orabove, or 61° C. or above, or 62° C. or above, or 63° C. or above, or64° C. or above, or 65° C. or above, 66° C. or above, or 67° C. orabove, or 68° C. or above, or 69° C. or above, or 70° C. or above. Insome embodiments, extreme annealing temperatures may be useful, such as30° C. to 80° C.

Split Primer

Many embodiments of the invention described herein relate to a splitprimer comprising a first target-specific section, a secondtarget-specific section, and an adaptor section positioned between thefirst target-specific section and the second target-specific section,and wherein the target-specific section is hybridizable to a targetsequence of a template DNA intended for amplification.

In some embodiments, the split primer further comprises a molecularindexing section comprising a molecule indexing sequence. The molecularindexing section can be, for example, positioned between the adaptorsection and one of the target-specific sections. The molecular indexingsection can be, for example, positioned at 3′ side of the adaptorsection. In some embodiments, the length of each molecular indexingsequence is about 1-20 bp, or about 2-15 bp, or about 3-10 bp, or about4-8 bp. When both a forward split primer and a reverse split primeraccording to the invention described herein are used for amplifying atarget locus of interests, the amplification product can include twomolecular indexing sequences, the combination of which allows even moreaccurate molecular counting than the use of a single molecular indexingsequence. In one embodiment, the molecule indexing sequence on eachsplit primer is a unique molecule indexing sequence. In anotherembodiment, the combination of molecule indexing sequences on each pairof split forward and reverse primers are unique.

In some embodiments, the adaptor section comprises a universal adaptorsequence for PCR amplification and/or sequencing.

Scheme C: FIG. 4 shows one scheme of the split primer in which anadaptor section positioned between a first target-specific section and asecond target-specific section. Both the first target-specific sectionand the second target-specific section are available for hybridizationto target sequences.

In other words, the target-specific section is split into two parts andthe universal adaptor sequence is placed in between. The molecularindexing sequences and adaptor sequences are protected after both endsof primers bind to target sequences. The split primer can beadvantageous in terms of reducing sequencing distance.

As shown in FIG. 12, a preferred embodiment of the split primeraccording to Scheme C comprises, from 5′ to 3′, first target-specificsection, an adaptor section, a molecular indexing section, secondtarget-specific section.

Split-Loopable Primer

Many embodiments of the invention described herein relate to asplit-loopable primer (a) comprising a first target-specific section, asecond target-specific section, and a stem-forming section positionedbetween the first target-specific section and the second target-specificsection, and an adaptor section, or (b) comprising a first adaptorsection, a second adaptor section, and a stem-forming section positionedbetween the first adaptor section and the second adaptor section, and atarget-specific section; and wherein the (first and/or second)target-specific section is hybridizable to a target sequence of atemplate DNA intended for amplification.

In some embodiments, the split-loopable primer further comprises amolecular indexing section comprising a molecule indexing sequence. Themolecular indexing section can be, for example, positioned between theadaptor section and the (second) target-specific sections. The molecularindexing section can be, for example, positioned at 3′ side of the(second) adaptor section. In some embodiments, the length of eachmolecular indexing sequence is about 1-20 bp, or about 2-15 bp, or about3-10 bp, or about 4-8 bp. When both a forward split-loopable primer anda reverse split-loopable primer according to the invention describedherein are used for amplifying a target locus of interests, theamplification product can include two molecular indexing sequences, thecombination of which allows even more accurate molecular counting thanthe use of a single molecular indexing sequence. In one embodiment, themolecule indexing sequence on each split-loopable primer is a uniquemolecule indexing sequence. In another embodiment, the combination ofmolecule indexing sequences on each pair of split-loopable forward andreverse primers are unique.

In some embodiments, the (first and/or second) adaptor section comprisesa universal adaptor sequence for PCR amplification and/or sequencing.

Scheme D: FIG. 13 shows one scheme of the split-loopable primercomprising a first target-specific section, a second target-specificsection, and a stem-forming section positioned between the firsttarget-specific section and the second target-specific section, and anadaptor section, wherein the stem-forming section is hybridizable to thesecond target-specific section to form a stem structure. When the secondtarget-specific section is protected in the stem structure, the firsttarget-specific section is available for initiating targethybridization.

In some embodiments, hybridization between the stem-forming section andthe second target-specific section forms a loop that comprises theadaptor section and the molecule indexing sequence. The stem loop isdesigned to protect the molecule indexing sequence and the adaptorsequence from spurious interactions. The stem in the 3′-end alsoprevents primers from non-target specific extension (i.e. primerdimers).

In some embodiments, the split-loopable primer further comprises one ormore mismatched nucleotides at 3′-terminus of the second target-specificsection that are not hybridizable to the stem-forming section. In someembodiments, the split-loopable primer comprises 1, 2, 3, 4, or 5mismatched nucleotides at 3′-terminus to prevent A-tailing.Alternatively or additionally, the 5′-end of the stem-forming sectionmay comprise one or more mismatched nucleotides that are nothybridizable to the second target-specific section. In some embodiments,the split-loopable primer comprises 1, 2, 3, 4, or 5 mismatchednucleotides at 5′-end of the stem-forming section.

As shown in FIG. 13, a preferred embodiment of the split-loopable primeraccording to Scheme D comprises, from 5′ to 3′, first target-specificsection, one or more mismatched nucleotides, a stem-forming section, anadaptor section, a molecular indexing section, and secondtarget-specific section, wherein the stem-forming section is the reversecomplement of part of the second target-specific section.

In some embodiments, the size of the stem structure formed between thestem-forming section and the second target-specific section is about5-20 bp, or about 5-10 bp, or about 10-15 bp, or about 15-20 bp.

In some embodiments, the first target-specific section is longer thanthe second target-specific section. In some embodiments, the secondtarget-specific section is longer than the first target-specificsection.

In some embodiments, at least 30%, or at least 40%, or at least 50%, orat least 60%, or at least 70%, or at least 80%, or at least 90% of thesecond target-specific section is hybridizable to the stem-formingregion and capable of forming a stem.

In some embodiments, the split-loopable primer according to Scheme D hasa preferred annealing temperature and a melting temperature for PCRreaction, wherein the stem-forming section and the secondtarget-specific section form the stem structure at the preferredannealing temperature or below and do not form the stem structure at themelting temperature or above. In some embodiments, the preferredannealing temperature is 60° C. or less, 59° C. or less, or 58° C. orless, or 57° C. or less, or 56° C. or less, or 55° C. or less, 54° C. orless, or 53° C. or less, or 52° C. or less, or 51° C. or less, or 50° C.or less. In some embodiments, the melting temperature is 60° C. orabove, or 61° C. or above, or 62° C. or above, or 63° C. or above, or64° C. or above, or 65° C. or above, 66° C. or above, or 67° C. orabove, or 68° C. or above, or 69° C. or above, or 70° C. or above. Insome embodiments, extreme annealing temperatures may be useful, such as30° C. to 80° C.

Scheme E: FIG. 13 shows another scheme of the split-loopable primercomprising a first adaptor section, a second adaptor section, and astem-forming section positioned between the first adaptor section andthe second adaptor section, and a target-specific section comprising a5′-portion and a 3′-portion, wherein the stem-forming section ishybridizable to the 3′-portion of the target-specific section to form astem structure. When the 3′-portion of the target-specific section isprotected in the stem structure, the 5′-portion of the target-specificsection is available for initiating target hybridization.

In some embodiments, hybridization between the stem-forming section andthe 3′-portion of the target-specific section forms a loop thatcomprises the second adaptor section, the molecule indexing sequence,and the 5′-portion of the target-specific section. The stem loop isdesigned to protect the molecule indexing sequence and the secondadaptor sequence from spurious interactions. The stem in the 3′-end alsoprevents primers from non-target specific extension (i.e. primerdimers).

In some embodiments, the split-loopable primer further comprises one ormore mismatched nucleotides at 3′-terminus of the target-specificsection that are not hybridizable to the stem-forming section. In someembodiments, the split-loopable primer comprises 1, 2, 3, 4, or 5mismatched nucleotides at 3′-terminus to prevent A-tailing.Alternatively or additionally, the 5′-end of the stem-forming sectionmay comprise one or more mismatched nucleotides that are nothybridizable to the target-specific section. In some embodiments, thesplit-loopable primer comprises 1, 2, 3, 4, or 5 mismatched nucleotidesat 5′-end of the stem-forming section

As shown in FIG. 13, a preferred embodiment of the split-loopable primeraccording to Scheme E comprises, from 5′ to 3′, first adaptor section,one or more mismatched nucleotides, a stem-forming section, secondadaptor section, a molecular indexing section, and a target-specificsection, wherein the stem-forming section is the reverse complement ofthe 3′-portion of the target-specific section.

In some embodiments, the size of the stem structure formed between thestem-forming section and the 3′-portion of the target-specific sectionis about 5-20 bp, or about 5-10 bp, or about 10-15 bp, or about 15-20bp.

In some embodiments, the first adaptor section is longer than the secondadaptor section. In some embodiments, the second adaptor section islonger than the first adaptor section.

In some embodiments, the split-loopable primer according to Scheme E hasa preferred annealing temperature for PCR reaction and a meltingtemperature, wherein the stem-forming section and the 3′-portion of thetarget-specific section form the stem structure at the preferredannealing temperature or below and do not form the stem structure at themelting temperature or above. In some embodiments, the preferredannealing temperature is 60° C. or less, 59° C. or less, or 58° C. orless, or 57° C. or less, or 56° C. or less, or 55° C. or less, 54° C. orless, or 53° C. or less, or 52° C. or less, or 51° C. or less, or 50° C.or less. In some embodiments, the melting temperature is 60° C. orabove, or 61° C. or above, or 62° C. or above, or 63° C. or above, or64° C. or above, or 65° C. or above, 66° C. or above, or 67° C. orabove, or 68° C. or above, or 69° C. or above, or 70° C. or above. Insome embodiments, extreme annealing temperatures may be useful, such as30° C. to 80° C.

Primer Composition

Further embodiments of the invention described herein relate to a primercomposition comprising the loopable primers, split primers, and/orsplit-loopable primers described herein.

In some embodiments, the primer composition comprises at least a forwardloopable primer and a reverse loopable primer that target the same locusof interest for amplification. In some embodiments, both the forwardloopable primer and the reverse loopable primer correspond to Scheme Ashown in FIG. 2. In some embodiments, both the forward loopable primerand the reverse loopable primer correspond to Scheme B shown in FIG. 3.

In some embodiments, the primer composition comprises at least a forwardsplit primer and a reverse split primer that target the same locus ofinterest for amplification. In some embodiments, both the forward splitprimer and the reverse loopable split correspond to Scheme C shown inFIG. 3.

In some embodiments, the primer composition comprises at least a forwardsplit-loopable primer and a reverse split-loopable primer that targetthe same locus of interest for amplification. In some embodiments, boththe forward split-loopable primer and the reverse split-loopable primercorrespond to Scheme E shown in FIG. 13. In some embodiments, both theforward split-loopable primer and the reverse split-loopable primercorrespond to Scheme E shown in FIG. 13.

In some embodiments, the composition comprises at least 50, at least100, at least 200, at least 500, at least 1,000, at least 2,000, atleast 5,000, or at least 10,000 different loopable primers. In someembodiments, the composition comprises at least 50, at least 100, atleast 200, at least 500, at least 1,000, at least 2,000, at least 5,000,or at least 10,000 different pairs of forward and reverse loopableprimers.

In some embodiments, the composition comprises at least 50, at least100, at least 200, at least 500, at least 1,000, at least 2,000, atleast 5,000, or at least 10,000 different loopable primers eachcomprising a different stem-forming section. In some embodiments, thecomposition comprises at least 50, at least 100, at least 200, at least500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000different loopable primers each comprising a different molecularindexing sequence. In some embodiments, the composition comprises atleast 200, at least 500, at least 1,000, at least 2,000, at least 5,000,at least 10,000, at least 20,000, at least 50,000, or at least 100,000different loopable primers each comprising a different combination ofthe stem-forming section and the molecular indexing sequence.

In some embodiments, the composition comprises at least 50, at least100, at least 200, at least 500, at least 1,000, at least 2,000, atleast 5,000, or at least 10,000 different split primers. In someembodiments, the composition comprises at least 50, at least 100, atleast 200, at least 500, at least 1,000, at least 2,000, at least 5,000,or at least 10,000 different pairs of forward and reverse split primers.

In some embodiments, the composition comprises at least 50, at least100, at least 200, at least 500, at least 1,000, at least 2,000, atleast 5,000, or at least 10,000 different split primers each comprisinga different target-specific section. In some embodiments, thecomposition comprises at least 50, at least 100, at least 200, at least500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000different split primers each comprising a different molecular indexingsequence. In some embodiments, the composition comprises at least 200,at least 500, at least 1,000, at least 2,000, at least 5,000, at least10,000, at least 20,000, at least 50,000, or at least 100,000 differentsplit primers each comprising a different combination of thetarget-specific section and the molecular indexing sequence.

In some embodiments, the composition comprises at least 50, at least100, at least 200, at least 500, at least 1,000, at least 2,000, atleast 5,000, or at least 10,000 different split-loopable primers. Insome embodiments, the composition comprises at least 50, at least 100,at least 200, at least 500, at least 1,000, at least 2,000, at least5,000, or at least 10,000 different pairs of forward and reversesplit-loopable primers.

In some embodiments, the composition comprises at least 50, at least100, at least 200, at least 500, at least 1,000, at least 2,000, atleast 5,000, or at least 10,000 different split-loopable primers eachcomprising a different stem-forming section. In some embodiments, thecomposition comprises at least 50, at least 100, at least 200, at least500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000different split-loopable primers each comprising a different molecularindexing sequence. In some embodiments, the composition comprises atleast 200, at least 500, at least 1,000, at least 2,000, at least 5,000,at least 10,000, at least 20,000, at least 50,000, or at least 100,000different split-loopable primers each comprising a different combinationof the stem-forming section and the molecular indexing sequence.

Methods for Amplification of Nucleic Acids

Further embodiments of the invention described herein relate to a methodfor amplifying a target locus of interest from a template DNA,comprising at least two pre-amplification cycles using the loopableprimer described above or the split primer described above or thesplit-loopable primer described above, wherein each pre-amplificationcycle comprises annealing the primer to the template DNA orpre-amplification product thereof and elongating the annealed primer.

In some embodiments, the method comprises at least three, at least four,at least five, at least ten, or up to fifteen, or up to ten, or up toseven, or up to five pre-amplification cycles.

In some embodiments, each pre-amplification cycle comprises annealing atleast a forward loopable primer and a reverse loopable primer thattarget the same locus of interest to the template DNA orpre-amplification product thereof, and elongating the annealed forwardloopable primer and the annealed reverse loopable primer. In someembodiments, both the forward loopable primer and the reverse loopableprimer correspond to Scheme A shown in FIG. 2. In some embodiments, boththe forward loopable primer and the reverse loopable primer correspondto Scheme B shown in FIG. 3.

In some embodiments, each pre-amplification cycle comprises annealing atleast a forward split primer and a reverse split primer that target thesame locus of interest to the template DNA or pre-amplification productthereof, and elongating the annealed forward split primer and theannealed reverse split primer. In some embodiments, both the forwardloopable primer and the reverse loopable primer correspond to Scheme Cshown in FIG. 4.

In some embodiments, each pre-amplification cycle comprises annealing atleast a forward split-loopable primer and a reverse split-loopableprimer that target the same locus of interest to the template DNA orpre-amplification product thereof, and elongating the annealed forwardsplit-loopable primer and the annealed reverse split-loopable primer. Insome embodiments, both the forward split-loopable primer and the reversesplit-loopable primer correspond to Scheme D shown in FIG. 13. In someembodiments, both the forward split-loopable primer and the reversesplit-loopable primer correspond to Scheme E shown in FIG. 13.

As shown in FIG. 10, when a pair of forward and reverse loopable primersaccording to Scheme A are used, the pre-amplification product cancomprise, for example, from 5′ to 3′, one or more mismatchednucleotides, first stem-forming section, first adaptor section, firstmolecular indexing section, amplified target sequences, second molecularindexing section, second adaptor section, second stem-forming section,and one or more mismatched nucleotides.

As shown in FIG. 11, when a pair of forward and reverse loopable primersaccording to Scheme B are used, the pre-amplification product cancomprise, for example, from 5′ to 3′, first stem-forming section, one ormore G/C nucleotides, first adaptor section, first molecular indexingsection, amplified target sequences, second molecular indexing section,second adaptor section, one or more G/C nucleotides, and secondstem-forming section.

As shown in FIG. 12, when a pair of forward and reverse split primersaccording to Scheme C are used, the pre-amplification product cancomprise, for example, from 5′ to 3′, 5′-target-specific sequences,first adaptor section, first molecular indexing section, amplifiedtarget sequences, second molecular indexing section, second adaptorsection, and 3′-target-specific sequences.

In some embodiments, the adaptor section comprises a universal adaptorsequence for PCR amplification, and wherein the method further comprisesa plurality of PCR cycles using one or more PCR primers hybridizable tothe universal adaptor sequence.

In some embodiments, the PCR primer comprises a sequencing adaptor fordownstream high-throughput sequencing of the PCR products. In someembodiments, the PCR primer comprises a sample barcode for pooling ofthe PCR products for further analysis.

In some embodiments, each pre-amplification cycle comprises annealing atleast 50, at least 100, at least 200, at least 500, at least 1,000, atleast 2,000, at least 5,000, or at least 10,000 different loopableprimers each comprising a different stem-forming section, to thetemplate DNA or pre-amplification product thereof. In some embodiments,each pre-amplification cycle comprises annealing at least 50, at least100, at least 200, at least 500, at least 1,000, at least 2,000, atleast 5,000, or at least 10,000 different loopable primers eachcomprising a different molecular indexing sequence, to the template DNAor pre-amplification product thereof. In some embodiments, eachpre-amplification cycle comprises annealing at least 200, at least 500,at least 1,000, at least 2,000, at least 5,000, at least 10,000, atleast 20,000, at least 50,000, or at least 100,000 different loopableprimers each comprising a different combination of the stem-formingsection and the molecular indexing sequence, to the template DNA orpre-amplification product thereof.

In some embodiments, each pre-amplification cycle comprises annealing atleast 50, at least 100, at least 200, at least 500, at least 1,000, atleast 2,000, at least 5,000, or at least 10,000 different pairs offorward and reverse loopable primers to the template DNA orpre-amplification product thereof.

In some embodiments, each pre-amplification cycle comprises annealing atleast 50, at least 100, at least 200, at least 500, at least 1,000, atleast 2,000, at least 5,000, or at least 10,000 different split primerseach comprising a different target-specific section, to the template DNAor pre-amplification product thereof. In some embodiments, eachpre-amplification cycle comprises annealing at least 50, at least 100,at least 200, at least 500, at least 1,000, at least 2,000, at least5,000, or at least 10,000 different split primers each comprising adifferent molecular indexing sequence, to the template DNA orpre-amplification product thereof. In some embodiments, eachpre-amplification cycle comprises annealing at least 200, at least 500,at least 1,000, at least 2,000, at least 5,000, at least 10,000, atleast 20,000, at least 50,000, or at least 100,000 split primers eachcomprising a different combination of the target-specific section andthe molecular indexing sequence, to the template DNA orpre-amplification product thereof.

In some embodiments, each pre-amplification cycle comprises annealing atleast 50, at least 100, at least 200, at least 500, at least 1,000, atleast 2,000, at least 5,000, or at least 10,000 different pairs offorward and reverse split primers to the template DNA orpre-amplification product thereof.

In some embodiments, each pre-amplification cycle comprises annealing atleast 50, at least 100, at least 200, at least 500, at least 1,000, atleast 2,000, at least 5,000, or at least 10,000 different split-loopableprimers each comprising a different stem-forming section, to thetemplate DNA or pre-amplification product thereof. In some embodiments,each pre-amplification cycle comprises annealing at least 50, at least100, at least 200, at least 500, at least 1,000, at least 2,000, atleast 5,000, or at least 10,000 different split-loopable primers eachcomprising a different molecular indexing sequence, to the template DNAor pre-amplification product thereof. In some embodiments, eachpre-amplification cycle comprises annealing at least 200, at least 500,at least 1,000, at least 2,000, at least 5,000, at least 10,000, atleast 20,000, at least 50,000, or at least 100,000 differentsplit-loopable primers each comprising a different combination of thestem-forming section and the molecular indexing sequence, to thetemplate DNA or pre-amplification product thereof.

In some embodiments, each pre-amplification cycle comprises annealing atleast 50, at least 100, at least 200, at least 500, at least 1,000, atleast 2,000, at least 5,000, or at least 10,000 different pairs offorward and reverse split-loopable primers to the template DNA orpre-amplification product thereof.

In some embodiments, the loopable primer according to Scheme A has apreferred annealing temperature for PCR reaction and a meltingtemperature, wherein the stem-forming section and the 3′-portion of thetarget-specific section form the stem structure at the preferredannealing temperature or below and do not form the stem structure at themelting temperature or above, and wherein the annealing temperature forthe pre-amplification cycles is at or below the preferred annealingtemperature (e.g., 60° C. or less, 59° C. or less, or 58° C. or less, or57° C. or less, or 56° C. or less, or 55° C. or less, 54° C. or less, or53° C. or less, or 52° C. or less, or 51° C. or less, or 50° C. orless).

In some embodiments, the loopable primer according to Scheme B has apreferred annealing temperature for PCR reaction and a meltingtemperature, wherein the stem-forming section and the 5′-portion of thetarget-specific section form the stem structure at the preferredannealing temperature or below and do not form the stem structure at themelting temperature or above, and wherein the annealing temperature forthe pre-amplification cycles is at or below the preferred annealingtemperature (e.g., 60° C. or less, 59° C. or less, or 58° C. or less, or57° C. or less, or 56° C. or less, or 55° C. or less, 54° C. or less, or53° C. or less, or 52° C. or less, or 51° C. or less, or 50° C. orless).

In some embodiments, the split-loopable primer according to Scheme D hasa preferred annealing temperature for PCR reaction and a meltingtemperature, wherein the stem-forming section and the secondtarget-specific section form the stem structure at the preferredannealing temperature or below and do not form the stem structure at themelting temperature or above, and wherein the annealing temperature forthe pre-amplification cycles is at or below the preferred annealingtemperature (e.g., 60° C. or less, 59° C. or less, or 58° C. or less, or57° C. or less, or 56° C. or less, or 55° C. or less, 54° C. or less, or53° C. or less, or 52° C. or less, or 51° C. or less, or 50° C. orless).

In some embodiments, the split-loopable primer according to Scheme E hasa preferred annealing temperature for PCR reaction and a meltingtemperature, wherein the stem-forming section and the 5′-portion of thetarget-specific section form the stem structure at the preferredannealing temperature or below and do not form the stem structure at themelting temperature or above, and wherein the annealing temperature forthe pre-amplification cycles is at or below the preferred annealingtemperature (e.g., 60° C. or less, 59° C. or less, or 58° C. or less, or57° C. or less, or 56° C. or less, or 55° C. or less, 54° C. or less, or53° C. or less, or 52° C. or less, or 51° C. or less, or 50° C. orless).

In other embodiments, the annealing temperature for thepre-amplification cycles can be below 50° C., or below 40° C., or below30° C., or below 20° C., or above 60° C., or above 65° C., or above 70°C. In other embodiments, extreme annealing temperatures may be usefulfor the pre-amplification cycles, such as 30° C. to 80° C.

Kits for Amplification of Nucleic Acids

Further embodiments of the invention described herein relate to a kitfor amplifying a target locus of interest from a template DNA,comprising a loopable primer described above or a split primer describedabove or a split-loopable primer described above.

In some embodiments, the kit comprises at least a forward loopableprimer and a reverse loopable primer that target the same locus ofinterest for amplification. In some embodiments, the kit comprises atleast a forward split primer and a reverse split primer that target thesame locus of interest for amplification. In some embodiments, the kitcomprises at least a forward split-loopable primer and a reversesplit-loopable primer that target the same locus of interest foramplification.

In some embodiments, the kit further comprises a polymerase forelongating the loopable primer or the split primer or the split-loopableprimer during the pre-amplification cycles.

In some embodiments, the kit further comprises a protease forinactivating the aforementioned polymerase upon completion of thepre-amplification cycles.

In some embodiments, the kit further comprises one or more PCR primershybridizable to the universal adaptor sequence in the adaptor section ofthe loopable primer or the split primer or the split-loopable primer. Insome embodiments, the PCR primer comprises a sequencing adaptor fordownstream high-throughput sequencing of the PCR products. In someembodiments, the PCR primer comprises a sample barcode for pooling ofthe PCR products for further analysis.

Applications

The loopable primer of Scheme A (3′-target-StemLoop) that hides/protectsthe universal adapter sequences and the MIT sequences improves assayspecificity by suppressing primer dimers and non-specific binding inhigh multiplex PCR. Accordingly, the loopable primer of Scheme A isparticularly useful for the following applications:

Copy number variant detection (CNV, aneuploidies, microdeletions, etc):With each DNA fragments product attached to a unique (or a uniquecombination) of molecular index tags, it allows tracking of the numberof fragments in a sample of a specific locus (sequence of amplicon).

PCR-error removal, real mutation detection: By using MIT barcodes, PCRartifacts, such as sequence changes generated by polymerase errors thatare not present in the original molecules can be identified andseparated from the real variants/mutations present in the originalmolecules.

PCR tiling: The stem in the 3′-end of the primers prevent primer dimerformation, which can be very useful for amplifying overlapping or tiledamplicons in a single multiplex PCR reaction.

Allele-specific amplification: Allele specific primers with the mutantbase position placed in the stem region (3′-end of primer) will inhibitthe stem from opening with the mismatch wild type and thereby preventamplification of the wild type.

Mutant allele specific quantitative PCR (qPCR) and digital PCR (qPCR):Primers for mutation and wild type have different tag sequences that canbe detected by different fluorescent probes colors and other detectionmethods.

Additional embodiments of the invention described herein relate to amethod for determining copy number variation of a target locus ofinterest, comprising: pre-amplifying the target locus of interest from atemplate DNA using at least two pre-amplification cycles with one ormore loopable primers each comprising a target-specific section, anadaptor section, a molecular indexing section, and a stem-formingsection, wherein the target-specific section comprises a 5′-portion anda 3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure,wherein the adaptor section comprises a universal adaptor sequence forPCR amplification, and wherein the molecular indexing section comprisesa molecule indexing sequence; amplifying the pre-amplification productusing one or more PCR primers hybridizable to the universal adaptorsequence; and sequencing the amplification product to determine copynumber variation of the target locus of interest using the moleculeindexing sequence.

Additional embodiments of the invention described herein relate to amethod for determining fetal aneuploidy, comprising: pre-amplifying aplurality of target loci of interest of one or more chromosomes fromcell-free DNA isolated from a maternal blood sample, using at least twopre-amplification cycles with a plurality of loopable primers eachcomprising a target-specific section, an adaptor section, a molecularindexing section, and a stem-forming section, wherein thetarget-specific section comprises a 5′-portion and a 3′-portion and thestem-forming section is hybridizable to the 3′-portion of thetarget-specific section to form a stem structure, wherein the adaptorsection comprises a universal adaptor sequence for PCR amplification,and wherein the molecular indexing section comprises a molecule indexingsequence; amplifying the pre-amplification product using one or more PCRprimers hybridizable to the universal adaptor sequence; and sequencingthe amplification product to determine fetal aneuploidy using themolecule indexing sequence.

Additional embodiments of the invention described herein relate to amethod for multiplex amplification, comprising: pre-amplifying one ormore target loci of interest from a template DNA using at least twopre-amplification cycles with at least a first loopable primer and asecond loopable primer each comprising a target-specific section, anadaptor section, and a stem-forming section, wherein the target-specificsection comprises a 5′-portion and a 3′-portion and the stem-formingsection is hybridizable to the 3′-portion of the target-specific sectionto form a stem structure, wherein the adaptor section comprises auniversal adaptor sequence for PCR amplification, and wherein the firstloopable primer and the second loopable primer comprise complementarysequences in their target-specific sections and are capable of forming aprimer dimer absent protection by the stem-forming section; andamplifying the pre-amplification product using one or more PCR primershybridizable to the universal adaptor sequence.

Additional embodiments of the invention described herein relate to amethod for allele-specific amplification, comprising: pre-amplifying oneor more target loci of interest from a template DNA using at least twopre-amplification cycles with a loopable primer comprising atarget-specific section, an adaptor section, and a stem-forming section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure,wherein the adaptor section comprises a universal adaptor sequence forPCR amplification, and wherein the loopable primer comprises an SNV orSNP allele in the 5′- or 3′-portion of the target-specific section; andamplifying the pre-amplification product using one or more PCR primershybridizable to the universal adaptor sequence.

Additional embodiments of the invention described herein relate to amethod for allele-specific quantitative PCR (qPCR), comprising:pre-amplifying one or more target loci of interest from a template DNAusing at least two pre-amplification cycles with at least a firstloopable primer and a second loopable primer each comprising atarget-specific section, an adaptor section, and a stem-forming section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure,wherein the adaptor section of the first loopable primer comprises auniversal adaptor sequence for PCR amplification and a firstprobe-specific sequence capable of binding to a first fluorescent probe,wherein the adaptor section of the second loopable primer comprises auniversal adaptor sequence for PCR amplification and a secondprobe-specific sequence capable of binding to a second fluorescentprobe, wherein the 5′- or 3′-portion of the target-specific section ofthe first loopable primer comprises a first SNV or SNP allele, andwherein the 5′- or 3′-portion of the target-specific section of thesecond loopable primer comprises a second SNV or SNP allele; amplifyingthe pre-amplification product using one or more PCR primers hybridizableto the universal adaptor sequence in the presence of the firstfluorescent probe and the second fluorescent probe; and detectingreal-time intensity of fluorescent signal from the first fluorescentprobe and the second fluorescent probe. Alternatively, the method forallele-specific qPCR does not require a pre-amplification step, andinstead comprises amplifying one or more target loci of interest from atemplate DNA using the first and second loopable primers in the presenceof the first and second fluorescent probes; and detecting real-timeintensity of fluorescent signal from the first and second fluorescentprobes.

Additional embodiments of the invention described herein relate to amethod for allele-specific digital PCR (dPCR), comprising:pre-amplifying one or more target loci of interest from a template DNAusing at least two pre-amplification cycles with at least a firstloopable primer and a second loopable primer each comprising atarget-specific section, an adaptor section, and a stem-forming section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure,wherein the adaptor section of the first loopable primer comprises auniversal adaptor sequence for PCR amplification and a firstprobe-specific sequence capable of binding to a first fluorescent probe,wherein the adaptor section of the second loopable primer comprises auniversal adaptor sequence for PCR amplification and a secondprobe-specific sequence capable of binding to a second fluorescentprobe, wherein the 5′- or 3′-portion of the target-specific section ofthe first loopable primer comprises a first SNV or SNP allele, andwherein the 5′- or 3′-portion of the target-specific section of thesecond loopable primer comprises a second SNV or SNP allele;partitioning the pre-amplification product into a plurality of reactionvolumes; amplifying the pre-amplification product in each reactionvolume using one or more PCR primers hybridizable to the universaladaptor sequence in the presence of the first fluorescent probe and thesecond fluorescent probe; and detecting presence or absence offluorescent signal from the first fluorescent probe and the secondfluorescent probe. Alternatively, the method for allele-specific dPCRdoes not require a pre-amplification step, and instead comprisespartitioning a sample into a plurality of reaction volumes; amplifyingone or more target loci of interest from a template DNA in each reactionvolume using the first and second loopable primers in the presence ofthe first and second fluorescent probes; and detecting presence orabsence of fluorescent signal from the first and second fluorescentprobes.

WORKING EXAMPLES Example 1: 2-Cycle Workflow

A proof-of-concept experiment was conducted to amplify sample DNA usingthe 2-cycle workflow as shown in FIG. 5. For each combination of primerscheme (FIG. 1), commercially available high fidelity enzyme mixes andDNA was prepared.

Master Mix initial vol [ul] final 1.6X Enzyme Mix 1.6 X 6.25 1 X 4XPrimer Mix 4 X 2.5 1 X DNA 1.25 1 X Final 10 *same formulation for allpolymerase tested.

MIT by Direct-PCR Reactions: Samples were cycled at the followingconditions. After 2 cycles, 20 μL of prepared Protease solution wasadded to the reaction and incubated at 65° C. for 15 minutes, followedby a 15 minute inactivation at 95° C.

DNA Primer Annealing Annealing PCR Polymerase Primers input [ng] Conc.[nM] Time [min] Temp [C] cycles* Condition MIT_F + MIT_R 15 ng 20 nM 6min 50° C 2 3 commercially available enzymes

Step # of Cycles Temperature Time Denaturation 1 98° C. 30 seconds Cycle2, 3 or 10 98° C. 10 seconds 50° C. 6 minutes 72° C. 30 seconds AddProtease 1 65° C. 15 minutes heating block in the hood Inactivation 95°C. 15 minutes Hold 1 4° C. infinite

Sequencing Barcoding Reactions: 10 μL of the 30 μL resultant volume wastaken into a Q5 barcoding reaction and cycled 35 times to full plateau.

reagent Stock Volume [ul] Final 2x Q5 Master 2 x 20.0 1 x Mix UniversalFBC 10 uM 1.6 0.4 uM primer Reverse RBC 5 uM 3.2 0.4 uM primerNuclease-free 5.2 water Barcoded DNA 10 (30 ul in total) Total volume 40

Step # of Cycles Temperature Time Hold 1 98° C. 3 minutes Cycle 35 98°C. 30 seconds 62.5° C. 30 seconds MAX mode 72° C. 30 seconds Hold 1 72°C. 2 minutes Hold 1 4° C. Infinite

Pooling and Purification: 2 μL of each sample were pooled together and50 μL of Pool was purified using Qiagen Qiaquick spin column.

Transfer 2 μL from each row of BAR plate(s) to strip tube. Cap and spin.Transfer contents into POOL_MIX1 tube Vortex POOL_MIX1 tubes for 10pulses of 2 seconds, quick spin for 5-10 seconds Transfer 50 μL ofPOOL_MIX1 to the new POOL_MIX2 tube. Discard POOL_MIX1 tubes, adhesiveseal and freeze the −BAR plate(s). Add 250 μL Qiagen PB Buffer. Add 5 μL3M NaOAc. Vortex for 10 pulses for 2 seconds and quick spin. Check thatthe color is yellow. Pipette pool into column. There should be 305 μL ofPool. Spin 14,000 × g for 1 minute. Discard flow-through and collectiontube. Replace with a new collection tube. Add 700 μL Qiagen PEbuffer(make sure EtOH was added). Spin at about 14,000 × g for 1 minute.Discard flow-through and collection tube. Replace with a new collectiontube. Spin at about 14,000 × g for 2 minute. Discard flow-through andcollection tube. Place column in the corresponding -POOL tube. Add 100μL Qiagen EB(elution buffer). Close cap and incubate at room temperaturefor 3 minutes. Spin at about 14,000 × g for 1 minute. If any residualelution buffer above column, pipette it to column and re-spin. Discardcolumn.

Sample was quantified by qPCR and sequenced.

As shown in FIG. 6, each of Scheme A, Scheme B, and Scheme C was able toamplify the target locus of interest with 2 pre-amplification cycles(followed by downstream PCR amplification using primers hybridizable tothe universal adaptor sequence), with scheme A showing the beston-target rate. As shown in FIG. 9, MIT counts were very consistentbetween replicate samples (Scheme A, 2 pre-amplification cycles).

Example 2: 3/10-Cycle Workflow

A proof-of-concept experiment was conducted to amplify sample DNA usingthe 3-cycle or 10-cycle workflow as shown in FIG. 7. For eachcombination of primer scheme (see FIG. 1), commercially available highfidelity enzyme mixes and DNA was prepared.

Master Mix initial vol [ul] final 1.6X Enzyme Mix 1.6 X 6.25 1 X 4XPrimer Mix 4 X 2.5 1 X DNA 1.25 1 X Final 10 *same formulation for allpolymerase tested.

MIT by Direct-PCR Reactions: Samples were cycled at the followingconditions. After 3 or 10 cycles, 20 μL of prepared Protease solutionwas added to the reaction and incubated at 65° C. for 15 minutes,followed by a 15 minute inactivation at 95° C.

DNA Primer Annealing Annealing PCR Polymerase Primers input [ng] Conc.[nM] Time [min] Temp [C] cycles* Condition MIT_F + MIT_R 15 ng 20 nM 6min 50° C 3 3 commercially 10 available enzymes

Step # of Cycles Temperature Time Denaturation 1 98° C. 30 seconds Cycle2, 3 or 10 98° C. 10 seconds 50° C. 6 minutes 72° C. 30 seconds AddProtease 1 65° C. 15 minutes heating block in the hood inactivation 195° C. 15 minutes Hold 1 4° C. infinite

Sequencing Barcoding Reactions: 10 μL of the 30 μL resultant volume wastaken into a Q5 barcoding reaction and cycled 35 times to full plateau.

reagent Stock Volume [ul] Final 2x Q5 Master 2 x 20.0 1 x Mix UniversalFBC 10 uM 1.6 0.4 uM primer Reverse RBC 5 uM 3.2 0.4 uM primerNuclease-free 5.2 water Barcoded DNA 10 (30 ul in total) Total volume 40

Step # of Cycles Temperature Time Hold 1 98° C. 3 minutes Cycle 35 98°C. 30 seconds 62.5° C. 30 seconds MAX mode 72° C. 30 seconds Hold 1 72°C. 2 minutes Hold 1 4° C. Infinite

Pooling and Purification: 2 μL of each sample were pooled together and50 μL of Pool was purified using Qiagen Qiaquick spin column.

Transfer 2 μL from each row of BAR plate(s) to strip tube. Cap and spin.Transfer contents into POOL_MIX1 tube Vortex POOL_MIX1 tubes for 10pulses of 2 seconds, quick spin for 5-10 seconds Transfer 50 μL ofPOOL_MIX1 to the new POOL_MIX2 tube. Discard POOL_MIX1 tubes, adhesiveseal and freeze the −BAR plate(s). Add 250 μL Qiagen PB Buffer. Add 5 μL3M NaOAc. Vortex for 10 pulses for 2 seconds and quick spin. Check thatthe color is yellow. Pipette pool into column. There should be 305 μL ofPool. Spin 14,000 × g for 1 minute. Discard flow-through and collectiontube. Replace with a new collection tube. Add 700 μL Qiagen PEbuffer(make sure EtOH was added). Spin at about 14,000 × g for 1 minute.Discard flow-through and collection tube. Replace with a new collectiontube. Spin at about 14,000 × g for 2 minute. Discard flow-through andcollection tube. Place column in the corresponding -POOL tube. Add 100μL Qiagen EB(elution buffer). Close cap and incubate at room temperaturefor 3 minutes. Spin at about 14,000 × g for 1 minute. If any residualelution buffer above column, pipette it to column and re-spin. Discardcolumn.

Sample was quantified by qPCR and sequenced.

As shown in FIG. 8, each of Scheme A, Scheme B, and Scheme C was able toamplify the target locus of interest with 3 or 10 pre-amplificationcycles (followed by downstream PCR amplification using primershybridizable to the universal adaptor sequence), with scheme A showingthe best on-target rate at 3 pre-amplification cycles and scheme Cshowing the best on-target rate at 10 pre-amplification cycles.

In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations, which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scopes ofthis invention.

What is claimed is:
 1. A composition comprising a primer that is: (a) aloopable primer comprising a target-specific section, an adaptorsection, and a stem-forming section, wherein the stem-forming section ishybridizable to a portion of the target-specific section to form a stemstructure, or (b) a split primer comprising a first target-specificsection, a second target-specific section, and an adaptor sectionpositioned between the first target-specific section and the secondtarget-specific section, or (c) a split-loopable primer comprising afirst target-specific section, a second target-specific section, and astem-forming section positioned between the first target-specificsection and the second target-specific section, and an adaptor section,or comprising a first adaptor section, a second adaptor section, and astem-forming section positioned between the first adaptor section andthe second adaptor section, and a target-specific section.
 2. Thecomposition of claim 1, wherein the primer is a loopable primer.
 3. Thecomposition of claim 2, wherein the adaptor section is positionedbetween the target-specific section and the stem-forming section, andwherein hybridization between the stem-forming section and the portionof the target-specific section forms a loop that comprises the adaptorsection.
 4. The composition of claim 3, wherein the adaptor section ispositioned at 5′ side of the target-specific section and 3′ side of thestem-forming section.
 5. The composition of claim 2, wherein the primerfurther comprises a molecular indexing section comprising a moleculeindexing sequence.
 6. The composition of claim 5, wherein the molecularindexing section is positioned between the target-specific section andthe adaptor section.
 7. The composition of claim 6, wherein themolecular indexing section is positioned at 5′ side of thetarget-specific section and 3′ side of the adaptor section, and whereinhybridization between the stem-forming section and the portion of thetarget-specific section forms a loop that comprises the adaptor sectionand the molecular indexing section.
 8. The composition of claim 2,wherein the target-specific section comprises a 5′-portion and a3′-portion, and wherein the stem-forming section is hybridizable to the3′-portion of the target-specific section.
 9. The composition of claim8, wherein hybridization between the stem-forming section and the3′-portion of the target-specific section forms a loop that comprisesthe adaptor section and the 5′-portion of the target-specific section.10. The composition of claim 8, wherein the loopable primer furthercomprises one or more mismatched nucleotides at 3′-terminus of thetarget-specific section that are not hybridizable to the stem-formingsection.
 11. The composition of claim 2, wherein the target-specificsection comprises a 5′-portion and a 3′-portion, and wherein thestem-forming section is hybridizable to a 5′-portion of thetarget-specific section.
 12. The composition of claim 11, whereinhybridization between the stem-forming section and the 5′-portion of thetarget-specific section forms a loop that comprises the adaptor sectionbut not the 3′-end portion of the target-specific section.
 13. Thecomposition of claim 11, wherein the loopable primer further comprisesone or more of G or C nucleotides positioned at the 5′-side of thetarget-specific section for stabilizing the stem structure with one ormore complementary G or C nucleotides optionally positioned at the3′-side of the stem-forming section.
 14. The composition of claim 2,comprising at least a forward loopable primer and a reverse loopableprimer that target the same locus of interest for amplification.
 15. Thecomposition of claim 1, wherein the primer is a split primer.
 16. Thecomposition of claim 15, wherein the primer further comprises amolecular indexing section comprising a molecule indexing sequence. 17.The composition of claim 16, wherein the molecular indexing section ispositioned between the adaptor section and one of the target-specificsection.
 18. The composition of claim 17, wherein the molecular indexingsection is positioned at 3′ side of the adaptor section.
 19. Thecomposition of claim 15, comprising at least a forward split primer anda reverse split primer that target the same locus of interest foramplification.
 20. The composition of claim 1, wherein the primer is asplit-loopable primer comprising a first target-specific section, asecond target-specific section, and a stem-forming section positionedbetween the first target-specific section and the second target-specificsection, and an adaptor section.
 21. The composition of claim 20,wherein the adaptor section is positioned between the stem-formingsection and the second target-specific section, wherein hybridizationbetween the stem-forming section and a portion of the secondtarget-specific section forms a loop that comprises the adaptor section.22. The composition of claim 21, wherein the adaptor section ispositioned at 5′ side of the second target-specific section and 3′ sideof the stem-forming section.
 23. The composition of claim 22, whereinthe split-loopable primer further comprises one or more mismatchednucleotides at 3′-terminus of the second target-specific section thatare not hybridizable to the stem-forming section.
 24. The composition ofclaim 20, wherein the split-loopable primer further comprises amolecular indexing section comprising a molecule indexing sequence. 25.The composition of claim 24, wherein the molecular indexing section ispositioned between the adaptor section and the second target-specificsection.
 26. The composition of claim 25, wherein the molecular indexingsection is positioned at 5′ side of the second target-specific sectionand 3′ side of the adaptor section, and wherein hybridization betweenthe stem-forming section and a portion of the second target-specificsection forms a loop that comprises the adaptor section and themolecular indexing section.
 27. The composition of claim 20, comprisingat least a forward split-loopable primer and a reverse split-loopableprimer that target the same locus of interest for amplification.
 28. Thecomposition of claim 1, wherein the primer is a split-loopable primercomprising a first adaptor section, a second adaptor section, and astem-forming section positioned between the first adaptor section andthe second adaptor section, and a target-specific section.
 29. Thecomposition of claim 28, wherein the second adaptor section ispositioned between the stem-forming section and the target-specificsection, wherein hybridization between the stem-forming section and aportion of the target-specific section forms a loop that comprises thesecond adaptor section.
 30. The composition of claim 29, wherein thesecond adaptor section is positioned at 5′ side of the target-specificsection and 3′ side of the stem-forming section.
 31. The composition ofclaim 28, wherein the split-loopable primer further comprises amolecular indexing section comprising a molecule indexing sequence. 32.The composition of claim 31, wherein the molecular indexing section ispositioned between the second adaptor section and the target-specificsection.
 33. The composition of claim 32, wherein the molecular indexingsection is positioned at 5′ side of the target-specific section and 3′side of the second adaptor section, and wherein hybridization betweenthe stem-forming section and a portion of the target-specific sectionforms a loop that comprises the second adaptor section and the molecularindexing section.
 34. The composition of claim 28, wherein thetarget-specific section comprises a 5′-portion and a 3′-portion, whereinthe stem-forming section is hybridizable to the 3′-portion of thetarget-specific section, and wherein hybridization between thestem-forming section and the 3′-portion of the target-specific sectionforms a loop that comprises the second adaptor section and the5′-portion of the target-specific section.
 35. The composition of claim34, wherein the split-loopable primer further comprises one or moremismatched nucleotides at 3′-terminus of the target-specific sectionthat are not hybridizable to the stem-forming section.
 36. Thecomposition of claim 28, comprising at least a forward split-loopableprimer and a reverse split-loopable primer that target the same locus ofinterest for amplification.
 37. The composition of claim 1, wherein theadaptor section comprises a universal adaptor sequence for PCRamplification and/or sequencing.
 38. The composition of claim 1,comprising at least 50 different primers each comprising a differentstem-forming section.
 39. The composition of claim 1, comprising atleast 50 different primers each comprising a different molecularindexing sequence.
 40. The composition of claim 1, comprising at least2,500 different primers each comprising a different combination of thestem-forming section and the molecular indexing sequence.
 41. A methodfor amplifying a target locus of interest from a template DNA,comprising at least two pre-amplification cycles using a primer that is:(a) a loopable primer comprising a target-specific section, an adaptorsection, and a stem-forming section, wherein the stem-forming section ishybridizable to a portion of the target-specific section to form a stemstructure, or (b) a split primer comprising a first target-specificsection, a second target-specific section, and an adaptor sectionpositioned between the first target-specific section and the secondtarget-specific section, or (c) a split-loopable primer comprising afirst target-specific section, a second target-specific section, and astem-forming section positioned between the first target-specificsection and the second target-specific section, or comprising a firstadaptor section, a second adaptor section, and a stem-forming sectionpositioned between the first adaptor section and the second adaptorsection; wherein each pre-amplification cycle comprises annealing theprimer to the template DNA or pre-amplification product thereof andelongating the annealed primer.
 42. The method of claim 41, wherein themethod comprises three or more pre-amplification cycles using theloopable primer or the split primer or the split-loopable primer. 43.The method of claim 41, wherein the method comprises five or morepre-amplification cycles using the loopable primer or the split primeror the split-loopable primer.
 44. The method of claim 41, wherein themethod comprises ten or fewer pre-amplification cycles using theloopable primer or the split primer or the split-loopable primer. 45.The method of claim 41, wherein the primer is a loopable primer.
 46. Themethod of claim 45, wherein the adaptor section is positioned betweenthe target-specific section and the stem-forming section, and whereinhybridization between the stem-forming section and the portion of thetarget-specific section forms a loop that comprises the adaptor section.47. The method of claim 46, wherein the adaptor section is positioned at5′ side of the target-specific section and 3′ side of the stem-formingsection.
 48. The method of claim 45, wherein the primer furthercomprises a molecular indexing section comprising a molecule indexingsequence.
 49. The method of claim 48, wherein the molecular indexingsection is positioned between the target-specific section and theadaptor section.
 50. The method of claim 49, wherein the molecularindexing section is positioned at 5′ side of the target-specific sectionand 3′ side of the adaptor section, and wherein hybridization betweenthe stem-forming section and the portion of the target-specific sectionforms a loop that comprises the adaptor section and the molecularindexing sequence.
 51. The method of claim 45, wherein thetarget-specific section comprises a 5′-portion and a 3′-portion, andwherein the stem-forming section is hybridizable to the 3′-portion ofthe target-specific section.
 52. The method of claim 51, whereinhybridization between the stem-forming section and the 3′-portion of thetarget-specific section forms a loop that comprises the adaptor sectionand the 5′-portion of the target-specific section.
 53. The method ofclaim 51, wherein the loopable primer further comprises one or moremismatched nucleotides at 3′-terminus of the target-specific sectionthat are not hybridizable to the stem-forming section.
 54. The method ofclaim 45, wherein the target-specific section comprises a 5′-portion anda 3′-portion, and wherein the stem-forming section is hybridizable to a5′-portion of the target-specific section.
 55. The method of claim 54,wherein hybridization between the stem-forming section and the5′-portion of the target-specific section forms a loop that comprisesthe adaptor section but not the 3′-end portion of the target-specificsection.
 56. The method of claim 54, wherein the loopable primer furthercomprises one or more of G or C nucleotides positioned at the 5′-side ofthe target-specific section for stabilizing the stem structure with oneor more complementary G or C nucleotides optionally positioned at the3′-side of the stem-forming section.
 57. The method of claim 45, whereineach pre-amplification cycle comprises annealing at least a forwardloopable primer and a reverse loopable primer that target the same locusof interest to the template DNA or pre-amplification product thereof,and elongating the annealed forward loopable primer and the annealedreverse loopable primer.
 58. The method of claim 41, wherein the primeris a split primer.
 59. The method of claim 58, wherein the primerfurther comprises a molecular indexing section comprising a moleculeindexing sequence.
 60. The method of claim 59, wherein the molecularindexing section is positioned between the adaptor section and one ofthe target-specific section.
 61. The method of claim 60, wherein themolecular indexing section is positioned at 3′ side of the adaptorsection.
 62. The method of claim 58, wherein each pre-amplificationcycle comprises annealing at least a forward split primer and a reversesplit primer that target the same locus of interest to the template DNAor pre-amplification product thereof, and elongating the annealedforward split primer and the annealed reverse split primer.
 63. Themethod of claim 41, wherein the primer is a split-loopable primercomprising a first target-specific section, a second target-specificsection, and a stem-forming section positioned between the firsttarget-specific section and the second target-specific section, and anadaptor section.
 64. The method of claim 63, wherein the adaptor sectionis positioned between the stem-forming section and the secondtarget-specific section, wherein hybridization between the stem-formingsection and a portion of the second target-specific section forms a loopthat comprises the adaptor section.
 65. The method of claim 64, whereinthe adaptor section is positioned at 5′ side of the secondtarget-specific section and 3′ side of the stem-forming section.
 66. Themethod of claim 65, wherein the split-loopable primer further comprisesone or more mismatched nucleotides at 3′-terminus of the secondtarget-specific section that are not hybridizable to the stem-formingsection.
 67. The method of claim 63, wherein the split-loopable primerfurther comprises a molecular indexing section comprising a moleculeindexing sequence.
 68. The method of claim 67, wherein the molecularindexing section is positioned between the adaptor section and thesecond target-specific section.
 69. The method of claim 68, wherein themolecular indexing section is positioned at 5′ side of the secondtarget-specific section and 3′ side of the adaptor section, and whereinhybridization between the stem-forming section and a portion of thesecond target-specific section forms a loop that comprises the adaptorsection and the molecular indexing section.
 70. The method of claim 63,wherein each pre-amplification cycle comprises annealing at least aforward split-loopable primer and a reverse split-loopable primer thattarget the same locus of interest to the template DNA orpre-amplification product thereof, and elongating the annealed forwardsplit-loopable primer and the annealed reverse split-loopable primer.71. The method of claim 41, wherein the primer is a split-loopableprimer comprising a first adaptor section, a second adaptor section, anda stem-forming section positioned between the first adaptor section andthe second adaptor section, and an target-specific section.
 72. Themethod of claim 71, wherein the second adaptor section is positionedbetween the stem-forming section and the target-specific section,wherein hybridization between the stem-forming section and a portion ofthe target-specific section forms a loop that comprises the secondadaptor section.
 73. The method of claim 72, wherein the second adaptorsection is positioned at 5′ side of the target-specific section and 3′side of the stem-forming section.
 74. The method of claim 71, whereinthe split-loopable primer further comprises a molecular indexing sectioncomprising a molecule indexing sequence.
 75. The method of claim 74,wherein the molecular indexing section is positioned between the secondadaptor section and the target-specific section.
 76. The method of claim75, wherein the molecular indexing section is positioned at 5′ side ofthe target-specific section and 3′ side of the second adaptor section,and wherein hybridization between the stem-forming section and a portionof the target-specific section forms a loop that comprises the secondadaptor section and the molecular indexing section.
 77. The method ofclaim 76, wherein the target-specific section comprises a 5′-portion anda 3′-portion, wherein the stem-forming section is hybridizable to the3′-portion of the target-specific section, and wherein hybridizationbetween the stem-forming section and the 3′-portion of thetarget-specific section forms a loop that comprises the second adaptorsection and the 5′-portion of the target-specific section.
 78. Themethod of claim 77, wherein the split-loopable primer further comprisesone or more mismatched nucleotides at 3′-terminus of the target-specificsection that are not hybridizable to the stem-forming section.
 79. Themethod of claim 71, wherein each pre-amplification cycle comprisesannealing at least a forward split-loopable primer and a reversesplit-loopable primer that target the same locus of interest to thetemplate DNA or pre-amplification product thereof, and elongating theannealed forward split-loopable primer and the annealed reversesplit-loopable primer.
 80. The method of claim 41, wherein the adaptorsection comprises a universal adaptor sequence for PCR amplification,and wherein the method further comprises a plurality of PCR cycles usingone or more PCR primers hybridizable to the universal adaptor sequence.81. The method of claim 80, wherein the one or more PCR primers eachcomprises a sequencing adaptor and/or a sample barcode.
 82. The methodof claim 41, wherein each pre-amplification cycle comprises annealing atleast 50 different primers each comprising a different stem-formingsection to the template DNA or pre-amplification product thereof. 83.The method of claim 41, wherein each pre-amplification cycle comprisesannealing at least 50 different primers each comprising a differentmolecular indexing sequence to the template DNA or pre-amplificationproduct thereof.
 84. The method of claim 41, wherein eachpre-amplification cycle comprises annealing at least 2,500 differentprimers each comprising a different combination of the stem-formingsection and the molecular indexing sequence to the template DNA orpre-amplification product thereof.
 85. A kit for amplifying a targetlocus of interest, comprising a primer that is: (a) a loopable primercomprising a target-specific section, an adaptor section, and astem-forming section, wherein the stem-forming section is hybridizableto a portion of the target-specific section to form a stem structure, or(b) a split primer comprising a first target-specific section, a secondtarget-specific section, and an adaptor section positioned between thefirst target-specific section and the second target-specific section, or(c) a split-loopable primer comprising a first target-specific section,a second target-specific section, and a stem-forming section positionedbetween the first target-specific section and the second target-specificsection, or comprising a first adaptor section, a second adaptorsection, and a stem-forming section positioned between the first adaptorsection and the second adaptor section.
 86. The kit of claim 85, furthercomprising a polymerase.
 87. The kit of claim 85, further comprising aprotease.
 88. The kit of claim 85, further comprising one or more PCRprimers hybridizable to the universal adaptor sequence.
 89. The kit ofclaim 88, wherein the one or more PCR primers each comprises asequencing adaptor and/or a sample barcode.
 90. The kit of claim 85,wherein the primer is a loopable primer.
 91. The kit of claim 90,wherein the adaptor section is positioned between the target-specificsection and the stem-forming section, and wherein hybridization betweenthe stem-forming section and the portion of the target-specific sectionforms a loop that comprises the adaptor section.
 92. The kit of claim91, wherein the adaptor section is positioned at 5′ side of thetarget-specific section and 3′ side of the stem-forming section.
 93. Thekit of claim 90, wherein the primer further comprises a molecularindexing section comprising a molecule indexing sequence.
 94. The kit ofclaim 93, wherein the molecular indexing section is positioned betweenthe target-specific section and the adaptor section.
 95. The kit ofclaim 94, wherein the molecular indexing section is positioned at 5′side of the target-specific section and 3′ side of the adaptor section,and wherein hybridization between the stem-forming section and theportion of the target-specific section forms a loop that comprises theadaptor section and the molecular indexing sequence.
 96. The kit ofclaim 90, wherein the target-specific section comprises a 5′-portion anda 3′-portion, and wherein the stem-forming section is hybridizable tothe 3′-portion of the target-specific section.
 97. The kit of claim 96,wherein hybridization between the stem-forming section and the3′-portion of the target-specific section forms a loop that comprisesthe adaptor section and the 5′-portion of the target-specific section.98. The kit of claim 96, wherein the loopable primer further comprisesone or more mismatched nucleotides at 3′-terminus of the target-specificsection that are not hybridizable to the stem-forming section.
 99. Thekit of claim 90, wherein the target-specific section comprises a5′-portion and a 3′-portion, and wherein the stem-forming section ishybridizable to a 5′-portion of the target-specific section.
 100. Thekit of claim 99, wherein hybridization between the stem-forming sectionand the 5′-portion of the target-specific section forms a loop thatcomprises the adaptor section but not the 3′-end portion of thetarget-specific section.
 101. The kit of claim 99, wherein the loopableprimer further comprises one or more of G or C nucleotides positioned atthe 5′-side of the target-specific section for stabilizing the stemstructure with one or more complementary G or C nucleotides positionedoptionally at the 3′-side of the stem-forming section.
 102. The kit ofclaim 90, comprising at least a forward loopable primer and a reverseloopable primer that target the same locus of interest foramplification.
 103. The kit of claim 85, wherein the primer is a splitprimer.
 104. The kit of claim 103, wherein the primer further comprisesa molecular indexing section comprising a molecule indexing sequence.105. The kit of claim 104, wherein the molecular indexing section ispositioned between the adaptor section and one of the target-specificsection.
 106. The kit of claim 105, wherein the molecular indexingsection is positioned at 3′ side of the adaptor section.
 107. The kit ofclaim 103, comprising at least a forward split primer and a reversesplit primer that target the same locus of interest for amplification.108. The kit of claim 85, wherein the primer is a split-loopable primercomprising a first target-specific section, a second target-specificsection, and a stem-forming section positioned between the firsttarget-specific section and the second target-specific section, and anadaptor section.
 109. The kit of claim 108, wherein the adaptor sectionis positioned between the stem-forming section and the secondtarget-specific section, wherein hybridization between the stem-formingsection and a portion of the second target-specific section forms a loopthat comprises the adaptor section.
 110. The kit of claim 109, whereinthe adaptor section is positioned at 5′ side of the secondtarget-specific section and 3′ side of the stem-forming section. 111.The kit of claim 110, wherein the split-loopable primer furthercomprises one or more mismatched nucleotides at 3′-terminus of thesecond target-specific section that are not hybridizable to thestem-forming section.
 112. The kit of claim 108, wherein thesplit-loopable primer further comprises a molecular indexing sectioncomprising a molecule indexing sequence.
 113. The kit of claim 112,wherein the molecular indexing section is positioned between the adaptorsection and the second target-specific section.
 114. The kit of claim113, wherein the molecular indexing section is positioned at 5′ side ofthe second target-specific section and 3′ side of the adaptor section,and wherein hybridization between the stem-forming section and a portionof the second target-specific section forms a loop that comprises theadaptor section and the molecular indexing section.
 115. The kit ofclaim 108, comprising at least a forward split-loopable primer and areverse split-loopable primer that target the same locus of interest foramplification.
 116. The kit of claim 85, wherein the primer is asplit-loopable primer comprising a first adaptor section, a secondadaptor section, and a stem-forming section positioned between the firstadaptor section and the second adaptor section, and a target-specificsection.
 117. The kit of claim 116, wherein the second adaptor sectionis positioned between the stem-forming section and the target-specificsection, wherein hybridization between the stem-forming section and aportion of the target-specific section forms a loop that comprises thesecond adaptor section.
 118. The kit of claim 117, wherein the secondadaptor section is positioned at 5′ side of the target-specific sectionand 3′ side of the stem-forming section.
 119. The kit of claim 116,wherein the split-loopable primer further comprises a molecular indexingsection comprising a molecule indexing sequence.
 120. The kit of claim119, wherein the molecular indexing section is positioned between thesecond adaptor section and the target-specific section.
 121. The kit ofclaim 120, wherein the molecular indexing section is positioned at 5′side of the target-specific section and 3′ side of the second adaptorsection, and wherein hybridization between the stem-forming section anda portion of the target-specific section forms a loop that comprises thesecond adaptor section and the molecular indexing section.
 122. The kitof claim 116, wherein the target-specific section comprises a 5′-portionand a 3′-portion, wherein the stem-forming section is hybridizable tothe 3′-portion of the target-specific section, and wherein hybridizationbetween the stem-forming section and the 3′-portion of thetarget-specific section forms a loop that comprises the second adaptorsection and the 5′-end portion of the target-specific section.
 123. Thekit of claim 122, wherein the split-loopable primer further comprisesone or more mismatched nucleotides at 3′-terminus of the target-specificsection that are not hybridizable to the stem-forming section.
 124. Thekit of claim 123, comprising at least a forward split-loopable primerand a reverse split-loopable primer that target the same locus ofinterest for amplification.
 125. The kit of claim 85, wherein theadaptor section comprises a universal adaptor sequence for PCRamplification and/or sequencing.
 126. The kit of claim 85, comprising apool of at least 50 different primers each comprising a differentstem-forming section.
 127. The kit of claim 85, comprising a pool of atleast 50 different primers each comprising a different molecularindexing sequence.
 128. The kit of claim 85, comprising a pool of atleast 2,500 different primers each comprising a different combination ofthe stem-forming section and the molecular indexing sequence.
 129. Amethod for determining copy number variation of a target locus ofinterest, comprising: pre-amplifying the target locus of interest from atemplate DNA using at least two pre-amplification cycles with: (a) oneor more loopable primers each comprising a target-specific section, anadaptor section, a molecular indexing section, and a stem-formingsection, wherein the target-specific section comprises a 5′-portion anda 3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the adaptor section and the molecular indexing sectionand the 5′-portion of the target-specific section, wherein the adaptorsection comprises a universal adaptor sequence for PCR amplification,and wherein the molecular indexing section comprises a molecule indexingsequence, (b) one or more split-loopable primers each comprising a firsttarget-specific section, a second target-specific section, astem-forming section positioned between the first target-specificsection and the second target-specific section, a molecular indexingsection, and an adaptor section, wherein the stem-forming section ishybridizable to a portion of the second target-specific section to forma stem structure and a loop comprising the adaptor section and themolecular indexing section, wherein the adaptor section comprises auniversal adaptor sequence for PCR amplification, and wherein themolecular indexing section comprises a molecule indexing sequence, or(c) one or more split-loopable primers each comprising a first adaptorsection, a second adaptor section, a stem-forming section positionedbetween the first adaptor section and the second adaptor section, amolecular indexing section, and a target-specific section, wherein thetarget-specific section comprises a 5′-portion and a 3′-portion and thestem-forming section is hybridizable to the 3′-portion of thetarget-specific section to form a stem structure and a loop comprisingthe second adaptor section and the molecular indexing section and the5′-portion of the target-specific section, wherein the first and/orsecond adaptor sections comprise a universal adaptor sequence for PCRamplification, and wherein the molecular indexing section comprises amolecule indexing sequence; amplifying the pre-amplification productusing one or more PCR primers hybridizable to the universal adaptorsequence; and sequencing the amplification product to determine copynumber variation of the target locus of interest using the moleculeindexing sequence.
 130. A method for determining fetal aneuploidy,comprising: pre-amplifying a plurality of target loci of interest of oneor more chromosomes from cell-free DNA isolated from a maternal bloodsample, using at least two pre-amplification cycles with: (a) aplurality of loopable primers each comprising a target-specific section,an adaptor section, a molecular indexing section, and a stem-formingsection, wherein the target-specific section comprises a 5′-portion anda 3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the adaptor section and the molecular indexing sectionand the 5′-portion of the target-specific section, wherein the adaptorsection comprises a universal adaptor sequence for PCR amplification,and wherein the molecular indexing section comprises a molecule indexingsequence, (b) a plurality of split-loopable primers each comprising afirst target-specific section, a second target-specific section, astem-forming section positioned between the first target-specificsection and the second target-specific section, a molecular indexingsection, and an adaptor section, wherein the stem-forming section ishybridizable to a portion of the second target-specific section to forma stem structure and a loop comprising the adaptor section and themolecular indexing section, wherein the adaptor section comprises auniversal adaptor sequence for PCR amplification, and wherein themolecular indexing section comprises a molecule indexing sequence, or(c) a plurality of split-loopable primers each comprising a firstadaptor section, a second adaptor section, a stem-forming sectionpositioned between the first adaptor section and the second adaptorsection, a molecular indexing section, and a target-specific section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the second adaptor section and the molecular indexingsection and the 5′-portion of the target-specific section, wherein thefirst and/or second adaptor sections comprise a universal adaptorsequence for PCR amplification, and wherein the molecular indexingsection comprises a molecule indexing sequence; amplifying thepre-amplification product using one or more PCR primers hybridizable tothe universal adaptor sequence; and sequencing the amplification productto determine fetal aneuploidy using the molecule indexing sequence. 131.A method for multiplex amplification, comprising: pre-amplifying one ormore target loci of interest from a template DNA using at least twopre-amplification cycles with: (a) at least a first loopable primer anda second loopable primer each comprising a target-specific section, anadaptor section, and a stem-forming section, wherein the target-specificsection comprises a 5′-portion and a 3′-portion and the stem-formingsection is hybridizable to the 3′-portion of the target-specific sectionto form a stem structure and a loop comprising the adaptor section andthe 5′-portion of the target-specific section, wherein the adaptorsection comprises a universal adaptor sequence for PCR amplification,(b) at least a first split-loopable primer and a second split-loopableprimer each comprising a first target-specific section, a secondtarget-specific section, a stem-forming section positioned between thefirst target-specific section and the second target-specific section,and an adaptor section, wherein the stem-forming section is hybridizableto a portion of the second target-specific section to form a stemstructure and a loop comprising the adaptor section, wherein the adaptorsection comprises a universal adaptor sequence for PCR amplification, or(c) at least a first split-loopable primer and a second split-loopableprimer each comprising a first adaptor section, a second adaptorsection, a stem-forming section positioned between the first adaptorsection and the second adaptor section, and a target-specific section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the second adaptor section and the 5′-portion of thetarget-specific section, wherein the first and/or second adaptorsections comprise a universal adaptor sequence for PCR amplification;and wherein the first primer and the second primer comprisecomplementary sequences in their target-specific sections and arecapable of forming a primer dimer absent protection by the stem-formingsection; and amplifying the pre-amplification product using one or morePCR primers hybridizable to the universal adaptor sequence.
 132. Amethod for allele-specific amplification, comprising: pre-amplifying oneor more target loci of interest from a template DNA using at least twopre-amplification cycles with: (a) a loopable primer comprising atarget-specific section, an adaptor section, and a stem-forming section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the adaptor section and the 5′-portion of thetarget-specific section, wherein the adaptor section comprises auniversal adaptor sequence for PCR amplification, and wherein theloopable primer comprises an SNV or SNP allele in the 3′-portion of thetarget-specific section, (b) a split-loopable primer comprising a firsttarget-specific section, a second target-specific section, astem-forming section positioned between the first target-specificsection and the second target-specific section, and an adaptor section,wherein the stem-forming section is hybridizable to a portion of thesecond target-specific section to form a stem structure and a loopcomprising the adaptor section, wherein the adaptor section comprises auniversal adaptor sequence for PCR amplification, and wherein thesplit-loopable primer comprises an SNV or SNP allele in thetarget-specific section, or (c) a split-loopable primer comprising afirst adaptor section, a second adaptor section, a stem-forming sectionpositioned between the first adaptor section and the second adaptorsection, and a target-specific section, wherein the target-specificsection comprises a 5′-portion and a 3′-portion and the stem-formingsection is hybridizable to the 3′-portion of the target-specific sectionto form a stem structure and a loop comprising the second adaptorsection and the 5′-portion of the target-specific section, wherein thefirst and/or second adaptor sections comprise a universal adaptorsequence for PCR amplification, and wherein the split-loopable primercomprises an SNV or SNP allele in the 3′-portion of the target-specificsection; and amplifying the pre-amplification product using one or morePCR primers hybridizable to the universal adaptor sequence.
 133. Amethod for allele-specific quantitative PCR (qPCR), comprising:pre-amplifying one or more target loci of interest from a template DNAusing at least two pre-amplification cycles with: (a) at least a firstloopable primer and a second loopable primer each comprising atarget-specific section, an adaptor section, and a stem-forming section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the adaptor section and the 5′-portion of thetarget-specific section, wherein the adaptor section of the firstloopable primer comprises a universal adaptor sequence for PCRamplification and a first probe-specific sequence capable of binding toa first fluorescent probe, wherein the adaptor section of the secondloopable primer comprises a universal adaptor sequence for PCRamplification and a second probe-specific sequence capable of binding toa second fluorescent probe, wherein the 5′- or 3′-portion of thetarget-specific section of the first loopable primer comprises a firstSNV or SNP allele, and wherein the 5′- or 3′-portion of thetarget-specific section of the second loopable primer comprises a secondSNV or SNP allele, (b) at least a first split-loopable primer and asecond split-loopable primer each comprising a first target-specificsection, a second target-specific section, a stem-forming sectionpositioned between the first target-specific section and the secondtarget-specific section, and an adaptor section, wherein thestem-forming section is hybridizable to a portion of the secondtarget-specific section to form a stem structure and a loop comprisingthe adaptor section, wherein the adaptor section of the firstsplit-loopable primer comprises a universal adaptor sequence for PCRamplification and a first probe-specific sequence capable of binding toa first fluorescent probe, wherein the adaptor section of the secondsplit-loopable primer comprises a universal adaptor sequence for PCRamplification and a second probe-specific sequence capable of binding toa second fluorescent probe, wherein the target-specific section of thefirst split-loopable primer comprises a first SNV or SNP allele, andwherein the target-specific section of the second split-loopable primercomprises a second SNV or SNP allele, or (c) at least a firstsplit-loopable primer and a second split-loopable primer each comprisinga first adaptor section, a second adaptor section, a stem-formingsection positioned between the first adaptor section and the secondadaptor section, and a target-specific section, wherein thetarget-specific section comprises a 5′-portion and a 3′-portion and thestem-forming section is hybridizable to the 3′-portion of thetarget-specific section to form a stem structure and a loop comprisingthe second adaptor section and the 5′-portion of the target-specificsection, wherein the first and/or second adaptor sections of the firstsplit-loopable primer comprise a universal adaptor sequence for PCRamplification and a first probe-specific sequence capable of binding toa first fluorescent probe, wherein the first and/or second adaptorsections of the second split-loopable primer comprise a universaladaptor sequence for PCR amplification and a second probe-specificsequence capable of binding to a second fluorescent probe, wherein the5′- or 3′-portion of the target-specific section of the firstsplit-loopable primer comprises a first SNV or SNP allele, and whereinthe 5′- or 3′-portion of the target-specific section of the secondsplit-loopable primer comprises a second SNV or SNP allele; amplifyingthe pre-amplification product using one or more PCR primers hybridizableto the universal adaptor sequence in the presence of the firstfluorescent probe and the second fluorescent probe; and detectingreal-time intensity of fluorescent signal from the first fluorescentprobe and the second fluorescent probe.
 134. A method forallele-specific digital PCR (dPCR), comprising: pre-amplifying one ormore target loci of interest from a template DNA using at least twopre-amplification cycles with: (a) at least a first loopable primer anda second loopable primer each comprising a target-specific section, anadaptor section, and a stem-forming section, wherein the target-specificsection comprises a 5′-portion and a 3′-portion and the stem-formingsection is hybridizable to the 3′-portion of the target-specific sectionto form a stem structure and a loop comprising the adaptor section andthe 5′-portion of the target-specific section, wherein the adaptorsection of the first loopable primer comprises a universal adaptorsequence for PCR amplification and a first probe-specific sequencecapable of binding to a first fluorescent probe, wherein the adaptorsection of the second loopable primer comprises a universal adaptorsequence for PCR amplification and a second probe-specific sequencecapable of binding to a second fluorescent probe, wherein the 5′- or3′-portion of the target-specific section of the first loopable primercomprises a first SNV or SNP allele, and wherein the 5′- or 3′-portionof the target-specific section of the second loopable primer comprises asecond SNV or SNP allele, (b) at least a first split-loopable primer anda second split-loopable primer each comprising a first target-specificsection, a second target-specific section, a stem-forming sectionpositioned between the first target-specific section and the secondtarget-specific section, and an adaptor section, wherein thestem-forming section is hybridizable to a portion of the secondtarget-specific section to form a stem structure and a loop comprisingthe adaptor section, wherein the adaptor section of the firstsplit-loopable primer comprises a universal adaptor sequence for PCRamplification and a first probe-specific sequence capable of binding toa first fluorescent probe, wherein the adaptor section of the secondsplit-loopable primer comprises a universal adaptor sequence for PCRamplification and a second probe-specific sequence capable of binding toa second fluorescent probe, wherein the target-specific section of thefirst split-loopable primer comprises a first SNV or SNP allele, andwherein the target-specific section of the second split-loopable primercomprises a second SNV or SNP allele, or (c) at least a firstsplit-loopable primer and a second split-loopable primer each comprisinga first adaptor section, a second adaptor section, a stem-formingsection positioned between the first adaptor section and the secondadaptor section, and a target-specific section, wherein thetarget-specific section comprises a 5′-portion and a 3′-portion and thestem-forming section is hybridizable to the 3′-portion of thetarget-specific section to form a stem structure and a loop comprisingthe second adaptor section and the 5′-portion of the target-specificsection, wherein the first and/or second adaptor sections of the firstsplit-loopable primer comprise a universal adaptor sequence for PCRamplification and a first probe-specific sequence capable of binding toa first fluorescent probe, wherein the first and/or second adaptorsections of the second split-loopable primer comprise a universaladaptor sequence for PCR amplification and a second probe-specificsequence capable of binding to a second fluorescent probe, wherein the5′- or 3′-portion of the target-specific section of the firstsplit-loopable primer comprises a first SNV or SNP allele, and whereinthe 5′- or 3′-portion of the target-specific section of the secondsplit-loopable primer comprises a second SNV or SNP allele; partitioningthe pre-amplification product into a plurality of reaction volumes;amplifying the pre-amplification product in each reaction volume usingone or more PCR primers hybridizable to the universal adaptor sequencein the presence of the first fluorescent probe and the secondfluorescent probe; and detecting presence or absence of fluorescentsignal from the first fluorescent probe and the second fluorescentprobe.
 135. A method for allele-specific quantitative PCR (qPCR),comprising: amplifying one or more target loci of interest from atemplate DNA, in the presence of the first fluorescent probe and thesecond fluorescent probe, using: (a) at least a first loopable primerand a second loopable primer each comprising a target-specific section,an adaptor section, and a stem-forming section, wherein thetarget-specific section comprises a 5′-portion and a 3′-portion and thestem-forming section is hybridizable to the 3′-portion of thetarget-specific section to form a stem structure and a loop comprisingthe adaptor section and the 5′-portion of the target-specific section,wherein the adaptor section of the first loopable primer comprises auniversal adaptor sequence for PCR amplification and a firstprobe-specific sequence capable of binding to a first fluorescent probe,wherein the adaptor section of the second loopable primer comprises auniversal adaptor sequence for PCR amplification and a secondprobe-specific sequence capable of binding to a second fluorescentprobe, wherein the 5′- or 3′-portion of the target-specific section ofthe first loopable primer comprises a first SNV or SNP allele, andwherein the 5′- or 3′-portion of the target-specific section of thesecond loopable primer comprises a second SNV or SNP allele, (b) atleast a first split-loopable primer and a second split-loopable primereach comprising a first target-specific section, a secondtarget-specific section, a stem-forming section positioned between thefirst target-specific section and the second target-specific section,and an adaptor section, wherein the stem-forming section is hybridizableto a portion of the second target-specific section to form a stemstructure and a loop comprising the adaptor section, wherein the adaptorsection of the first split-loopable primer comprises a universal adaptorsequence for PCR amplification and a first probe-specific sequencecapable of binding to a first fluorescent probe, wherein the adaptorsection of the second split-loopable primer comprises a universaladaptor sequence for PCR amplification and a second probe-specificsequence capable of binding to a second fluorescent probe, wherein thetarget-specific section of the first split-loopable primer comprises afirst SNV or SNP allele, and wherein the target-specific section of thesecond split-loopable primer comprises a second SNV or SNP allele, or(c) at least a first split-loopable primer and a second split-loopableprimer each comprising a first adaptor section, a second adaptorsection, a stem-forming section positioned between the first adaptorsection and the second adaptor section, and a target-specific section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the second adaptor section and the 5′-portion of thetarget-specific section, wherein the first and/or second adaptorsections of the first split-loopable primer comprise a universal adaptorsequence for PCR amplification and a first probe-specific sequencecapable of binding to a first fluorescent probe, wherein the firstand/or second adaptor sections of the second split-loopable primercomprise a universal adaptor sequence for PCR amplification and a secondprobe-specific sequence capable of binding to a second fluorescentprobe, wherein the 5′- or 3′-portion of the target-specific section ofthe first split-loopable primer comprises a first SNV or SNP allele, andwherein the 5′- or 3′-portion of the target-specific section of thesecond split-loopable primer comprises a second SNV or SNP allele; anddetecting real-time intensity of fluorescent signal from the firstfluorescent probe and the second fluorescent probe.
 136. A method forallele-specific digital PCR (dPCR), comprising: partitioning a samplecomprising template DNAs into a plurality of reaction volumes;amplifying one or more target loci of interest from a template DNA ineach reaction volume, in the presence of the first fluorescent probe andthe second fluorescent probe, using: (a) at least a first loopableprimer and a second loopable primer each comprising a target-specificsection, an adaptor section, and a stem-forming section, wherein thetarget-specific section comprises a 5′-portion and a 3′-portion and thestem-forming section is hybridizable to the 3′-portion of thetarget-specific section to form a stem structure and a loop comprisingthe adaptor section and the 5′-portion of the target-specific section,wherein the adaptor section of the first loopable primer comprises auniversal adaptor sequence for PCR amplification and a firstprobe-specific sequence capable of binding to a first fluorescent probe,wherein the adaptor section of the second loopable primer comprises auniversal adaptor sequence for PCR amplification and a secondprobe-specific sequence capable of binding to a second fluorescentprobe, wherein the 5′- or 3′-portion of the target-specific section ofthe first loopable primer comprises a first SNV or SNP allele, andwherein the 5′- or 3′-portion of the target-specific section of thesecond loopable primer comprises a second SNV or SNP allele, (b) atleast a first split-loopable primer and a second split-loopable primereach comprising a first target-specific section, a secondtarget-specific section, a stem-forming section positioned between thefirst target-specific section and the second target-specific section,and an adaptor section, wherein the stem-forming section is hybridizableto a portion of the second target-specific section to form a stemstructure and a loop comprising the adaptor section, wherein the adaptorsection of the first split-loopable primer comprises a universal adaptorsequence for PCR amplification and a first probe-specific sequencecapable of binding to a first fluorescent probe, wherein the adaptorsection of the second split-loopable primer comprises a universaladaptor sequence for PCR amplification and a second probe-specificsequence capable of binding to a second fluorescent probe, wherein thetarget-specific section of the first split-loopable primer comprises afirst SNV or SNP allele, and wherein the target-specific section of thesecond split-loopable primer comprises a second SNV or SNP allele, or(c) at least a first split-loopable primer and a second split-loopableprimer each comprising a first adaptor section, a second adaptorsection, a stem-forming section positioned between the first adaptorsection and the second adaptor section, and a target-specific section,wherein the target-specific section comprises a 5′-portion and a3′-portion and the stem-forming section is hybridizable to the3′-portion of the target-specific section to form a stem structure and aloop comprising the second adaptor section and the 5′-portion of thetarget-specific section, wherein the first and/or second adaptorsections of the first split-loopable primer comprise a universal adaptorsequence for PCR amplification and a first probe-specific sequencecapable of binding to a first fluorescent probe, wherein the firstand/or second adaptor sections of the second split-loopable primercomprise a universal adaptor sequence for PCR amplification and a secondprobe-specific sequence capable of binding to a second fluorescentprobe, wherein the 5′- or 3′-portion of the target-specific section ofthe first split-loopable primer comprises a first SNV or SNP allele, andwherein the 5′- or 3′-portion of the target-specific section of thesecond split-loopable primer comprises a second SNV or SNP allele; anddetecting presence or absence of fluorescent signal from the firstfluorescent probe and the second fluorescent probe.